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10.7554/elife.91422	Diversity in Notch ligand-receptor signaling interactions	<jats:p>The Notch signaling pathway uses families of ligands and receptors to transmit signals to nearby cells. These components are expressed in diverse combinations in different cell types, interact in a many-to-many fashion, both within the same cell (in cis) and between cells (in trans), and their interactions are modulated by Fringe glycosyltransferases. A fundamental question is how the strength of Notch signaling depends on which pathway components are expressed, at what levels, and in which cells. Here, we used a quantitative, bottom-up, cell-based approach to systematically characterize trans-activation, cis-inhibition, and cis-activation signaling efficiencies across a range of ligand and Fringe expression levels in two mammalian cell types. Each ligand (Dll1, Dll4, Jag1, and Jag2) and receptor variant (Notch1 and Notch2) analyzed here exhibited a unique profile of interactions, Fringe-dependence, and signaling outcomes. All four ligands were able to bind receptors in cis and in trans, and all ligands trans-activated both receptors, although Jag1-Notch1 signaling was substantially weaker than other ligand-receptor combinations. Cis-interactions were predominantly inhibitory, with the exception of the Dll1-and Dll4-Notch2 pairs, which exhibited cis-activation stronger than trans-activation. Lfng strengthened Delta-mediated trans-activation and weakened Jagged-mediated trans-activation for both receptors. Finally, cis-ligands showed diverse cis-inhibition strengths, which depended on the identity of the trans-ligand as well as the receptor. The map of receptor-ligand-Fringe interaction outcomes revealed here should help guide rational perturbation and control of the Notch pathway.</jats:p>	[ { "section_content": "The Notch signaling pathway controls stem cell differentiation and proliferation, plays key roles in numerous diseases, and represents a major drug target.It uses multiple membrane-bound ligands and receptors that interact with one another in a many-to-many fashion, as well as Fringe glycosyltransferases (Fringes) that modulate those interactions.In mammals, the Notch pathway consists of four receptors (Notch1-4), four canonical activating ligands (Dll1, Dll4, Jag1, and Jag2), at least one predominantly inhibitory ligand (Dll3), and non-canonical ligands (D 'Souza et al., 2008;Falix et al., 2012;Fiddes et al., 2018;Gera and Dighe, 2018;Ladi et al., 2005;Serth et al., 2015).Ligands and receptors interact both within the same cell (in cis) and between adjacent cells (in trans). Either configuration has the potential to activate or inhibit signaling (del Álamo et al., 2011;Nandagopal et al., 2019;Sprinzak et al., 2010).The level of signaling in a Notch-expressing cell generally depends on which ligand, receptor, and Fringe variants are expressed in the cell and its neighbors.These components are expressed in various combinations in different cell types, often including coexpression of ligands, receptors, and at least one Fringe enzyme (Granados et al., 2022).However, it remains difficult to predict signaling strength-how strongly a given cell will signal to another cellbased on the expression profiles of Notch pathway components.It similarly remains challenging to rationally and predictably perturb signaling for therapeutic and tissue engineering purposes. Transcriptional responses are sensitive to the amplitude and duration of Notch signaling (Falo-Sanjuan et al., 2019;Kuang et al., 2020;Nandagopal et al., 2018).In the canonical trans-activation mechanism, binding of ligands on one cell to receptors on an adjacent cell triggers ligand endocytosis, which generates mechanical strain on the receptor, exposing a metalloproteinase recognition site (Langridge and Struhl, 2017;Lovendahl et al., 2018).S2 cleavage by ADAM10 results in shedding of the receptor extracellular domain (NECD) and permits a subsequent S3 cleavage by γ-secretase to release the Notch intracellular domain (NICD).The free NICD directly translocates into the nucleus and binds cofactors MAML and RBPjκ to activate target genes.Downstream target genes respond similarly to NICD originating from the Notch1 or Notch2 receptors (Kraman and McCright, 2005;Liu et al., 2015;Liu et al., 2013).However, they respond differently to distinct concentrations of NICD.In pancreatic progenitors (Ninov et al., 2012) and central nervous system stem cells (Guentchev and McKay, 2006), complete Notch inhibition permitted differentiation, low levels of NICD promoted proliferation, and higher levels induced quiescence.Notch signaling amplitude can also influence the timing of developmental transitions, with higher NICD concentrations activating master transcription factors earlier than lower concentrations, depending on the target's enhancer architecture (Falo-Sanjuan et al., 2019). The Notch pathway provides numerous ways to tune signaling amplitude.Fringe enzymes can alter receptor-ligand binding and activation strengths, sometimes in opposite directions (Hicks et al., 2000;Kakuda et al., 2020;Kakuda and Haltiwanger, 2017).Upregulating expression of a ligand can suppress Notch signaling cell-autonomously, through a process termed cis-inhibition (Becam et al., 2010;Fiuza et al., 2010;Sprinzak et al., 2010;Thambyrajah et al., 2024), or trans-activate receptors on neighboring cells.In some cases, ligands can also activate signaling by receptors in the same cell (Nandagopal et al., 2019).Finally, growing evidence suggests that some ligands can inhibit signaling intercellularly ('trans-inhibition') by binding receptors strongly without activating them (Benedito et al., 2009;Golson et al., 2009;Luna-Escalante et al., 2018), as may be the case for Jag1 and Lunatic Fringe (Lfng)-modified Notch1 (Hicks et al., 2000;Kakuda and Haltiwanger, 2017).Thus, the Notch pathway architecture allows for receptor-ligand interactions to result in either activation or inhibition of signaling, in either cis or trans.However, for the majority of possible receptor-ligand interaction pairs, the relative activation and inhibition strengths have not been measured in both cis and trans orientations, making it difficult to predict signaling outcomes in natural contexts and in applications such as tissue engineering. Here, to address these challenges, we developed a set of engineered cell lines and coculture reporter assays that allow systematic characterization of trans-activation, cis-inhibition, and cis-activation efficiencies across a range of cis-ligand and Fringe expression levels.We focused on interactions among four ligands-Dll1, Dll4, Jag1, and Jag2-with two receptors-Notch1 and Notch2-and their modulation by Lfng.We verified that key features are consistent between two distinct cell lines: Chinese hamster ovary (CHO-K1) fibroblasts and C2C12 myoblasts.Each receptor and ligand had a unique profile of Lfng-dependent cis-and trans-interactions with other components.In trans, all ligands were capable of activating Notch1 and Notch2.However, Jag1 trans-activated Notch1 inefficiently, despite the strong Notch1-activating ability of recombinant Jag1 fragments.In cis, with competition from trans-activating ligands, Dll1 and Dll4 inhibited Notch1 and further activated Notch2 signaling, while Jagged ligands cis-inhibited both receptors.Lfng modulated most receptor-ligand interactions.It increased Jag1's inhibitory potential by strengthening binding but weakening activation of Notch1, as seen previously (Hicks et al., 2000;Kakuda and Haltiwanger, 2017;Taylor et al., 2014;Yang et al., 2005).In addition, Lfng potentiated trans-activation for Delta-Notch combinations, but attenuated Jagged-Notch signaling.It also had diverse receptor-and ligand-specific effects on cis-activation, which in general differed from those on trans-activation for Notch2 but not for Notch1.Together, this map of relative cis and trans receptor-ligand-Fringe interactions should be useful to explain Notch signaling behaviors in diverse developmental and physiological contexts, and guide more rational, targeted perturbation of Notch signaling activity. ", "section_name": "Introduction", "section_num": null }, { "section_content": "Engineered 'receiver' and 'sender' cell lines enable quantitative comparison of receptor-ligand-Fringe interactions To systematically analyze pairwise cis and trans receptor-ligand interactions, and their dependence on Fringe enzyme expression, we engineered a set of over 50 different stable cell lines (Key resources table).Collectively, they provide quantitative readouts of Notch signaling activity, receptor level, and ligand level.They also enable precise modulation of ligand expression.As a base cell line, we used CHO-K1 cells, which exhibit negligible endogenous expression levels of Notch receptors and ligands, and no endogenous Notch signaling activity (Supplementary file 1; Singh et al., 2018;Sprinzak et al., 2010).In this background, we constructed three types of cell lines, described below. First, to read out Notch signaling, we created monoclonal 'receiver' cells, similar to those described previously (LeBon et al., 2014;Nandagopal et al., 2019;Sprinzak et al., 2010;Figure 1A).These cells expressed a chimeric human Notch receptor, whose intracellular domain was replaced with a minimal Gal4 transcription factor ('Gal4esn', henceforth denoted Gal4).This coding sequence was followed by H2B-mTurq2, with an intervening ribosomal skipping T2A sequence, for cotranslational readout of receptor expression.This construct was stably integrated in the host cell genome using piggyBac transposition (Methods).Expression of the cotranslational mTurq2 reporter was broadly comparable between different receiver clones (Figure 1-figure supplement 1A), and correlated with surface receptor expression (Figure 1-figure supplement 1B).The cells also contained an insulated UAS promoter driving expression of H2B-mCitrine, such that mCitrine production reflects Notch activity. Second, to analyze same-cell (cis) ligand-receptor interactions, we used lentivirus to stably integrate each of the four activating human Notch ligands (Dll1, Dll4, Jag1, Jag2) into the receiver cells (Figure 1A).All ligand constructs contained a T2A cotranslational H2B-mCherry reporter for readout of expression.Ligand expression was controlled by the Tet-OFF system, allowing the use of the doxycycline analog 4-epi-tetracycline (4-epi-Tc) to titrate ligand levels.In these lines, varying the concentration of 4-epi-Tc tuned expression unimodally across two orders of magnitude (Figure 1B, Figure 1-figure supplement 2).To control for non-specific effects of ligand overexpression, we also constructed parallel negative control cell lines expressing H2B-mCherry or human nerve growth factor receptor (NGFR)-T2A-H2B-mCherry constructs in place of ligands.NGFR has been used in other Notch studies as a surface-detectable coexpression reporter or marker (Del Real and Rothenberg, 2013;Romero-Wolf et al., 2020;Sakata-Yanagimoto et al., 2008;Taghon et al., 2009).These cell lines enabled control and readout of cis-ligand levels. Third, we engineered a repertoire of 'sender' cell lines (Figure 1A).One set of these cell lines enabled inducible expression of each of the four ligands under control of the Tet-OFF system (Figure 1-figure supplement 3, Methods).These lines allowed control of ligand expression in cells without Notch receptors.A second set provided constitutive expression of each ligand at a variety of different levels (Figure 1C). Because cotranslational protein abundance is limited as a proxy for surface protein, this study focuses primarily on the relationship between signaling activity and the total amount of translated receptor or ligand.However, we note that high signaling activity need not, in general, directly correlate with surface expression levels; e.g., factors such as vimentin decrease basal surface of Jag1 but increase its activity (Antfolk et al., 2017). To limit combinatorial complexity, we focused on two essential receptors (Notch1 and Notch2), four canonical activating ligands (Dll1, Dll4, Jag1, and Jag2), and one Fringe (Lfng).We omitted Notch3, since Notch3 knockout mice are viable with minor vascular defects (Kitamoto et al., 2005;Krebs et al., 2003), and because Notch3 receptors are hypersensitive to activation during cell passaging, yielding elevated background signaling.We also omitted Notch4, which has no knockout phenotype in mice (Krebs et al., 2000) does not appear to activate in coculture with ligand-expressing cells (Groot et al., 2014;James et al., 2014;Lafkas et al., 2015), and has been suggested to inhibit Notch1 activation in cis (James et al., 2014).Among the best-studied ligands, we omitted Dll3, which is essential and believed to be purely cis-inhibitory (Bochter et al., 2022).Finally, we focused on Lunatic Fringe (Lfng), which we perturbed using siRNAs and transient plasmid transfection, because it is the only Fringe enzyme that is essential in mice (Evrard et al., 1998;Moran et al., 2009;Zhang et al., 2002), and its effects dominate over those of Radical and Manic Fringe (Rfng and Mfng, respectively) when coexpressed (Pennarubia et al., 2021).Together, these cell lines enabled systematic analysis of trans-activation, cis-activation, and cis-inhibition (Figure 1D, Methods). ", "section_name": "Results", "section_num": null }, { "section_content": "A fundamental question about the Notch system is how signaling strength depends on the identity of the interacting ligand and receptor, and expression of Fringe enzymes.Previous investigations reached conflicting conclusions about how strongly and even in what direction Fringes affect different ligand-receptor interactions (Hicks et al., 2000;Kakuda et al., 2020;Shimizu et al., 2001;Yang et al., 2005).They also focused on only a subset of essential ligand-receptor combinations, omitted analysis of Fringe dependence (Tveriakhina et al., 2018), and in some cases used plate-bound ligands rather than ligands expressed by cells (Kakuda et al., 2020). To address these gaps, we used a trans-activation assay (Figure 1D) to systematically measure trans-signaling across all eight receptor-ligand combinations, under controlled Fringe expression conditions (Methods).We first suppressed endogenous Rfng and Lfng expression via siRNA knockdown in receiver cells (Figure 1E; Figure 1-figure supplement 4A; Supplementary file 1).Approximately 16 hr later, we removed siRNAs and transfected plasmids encoding wild-type (wt) mouse Lfng or, as a negative control, a catalytically inactive mutant Lfng (D289E, denoted 'dLfng') (Luther et al., 2009).We also cotransfected a plasmid expressing infrared fluorescent protein (IFP2) as a transfection marker.After recovering for 16-20 hr post-transfection, we cocultured these receiver cells with an excess of sender cells, previously sorted into bins of stable ligand expression (Figure 1C).Finally, after 22-24 hr of coculture, we measured Notch activity in receivers by flow cytometry, gating on mTurq2, expression of a stably integrated UAS-H2B-mCitrine reporter (yellow).Some strains also contained a stably integrated ligand and reporter (second cartoon).These sender cell lines were constructed by integrating plasmids containing each of the four activating ligands fused to 2xFLAG, followed by T2A-H2B-mCherry for cotranslational readout of ligand expression, either under Tet-OFF control (to allow induction by 4-epiTc) or expressed from the constitutive CBh promoter.(B) Example distributions of Tet-OFF inducible ligand expression in CHO-K1 cells, read out by fluorescence of a cotranslational H2B-mCherry (A.U.).Black histograms are CHO-K1 wild-type cells (NCC = no color control).Dotted gray vertical lines mark binning windows used in some analyses (see also Methods).Data points in the gray-shaded region were omitted to avoid overexpression artifacts (see Methods).(C) Single-cell histograms (kernel density estimates) of stably expressed ligand levels (read out by cotranslational H2B-mCherry, A.U.) in the CHO-K1 sender populations used for trans-activation assay experiments.n denotes number of replicates per plot.Sender populations are named with the ligand expressed and a population identifier (e.g.'L4B').(D) Schematics of the main assays used in this work.Each panel shows the cocultured cell types and their relative population sizes (majority or minority).In the cis-+trans-activation assay, dotted lines indicate alternative receptor interactions.See also Methods.Dot-and-arrow icons are used to identify assays in subsequent figures.(E) Experimental workflow for cell culture experiments with flow cytometry readout.Ligand expression is preinduced in either sender or receiver cells by reducing the 4-epi-Tc concentration in the culture medium to induce ligand expression to a given level.Receivers are incubated in the Notch signaling inhibitor DAPT to prevent reporter activation during this preinduction phase.Prepared cells, which may also undergo siRNA knockdown and/or plasmid transfection during the ligand preinduction phase, are replated without DAPT according to the chosen experimental scheme in (D) and allowed to signal for 22-24 hr before cells are detached and analyzed by flow cytometry.(F-G) Data plots show example flow cytometry data processing.Gates used in data processing are shown as dashed lines.(F) Senders and receivers are separated computationally in the flow cytometry data in a 2D plane of cell size (side scatter, A.U.) vs. cotranslational receptor expression (mTurquoise2, A.U.).(G) Plasmid-transfected cells are gated on fluorescence levels of a cotransfected infrared fluorescent protein (IFP2, A.U.).Data points in gray-shaded regions were discarded (Methods). The online version of this article includes the following figure supplement(s) for figure 1: To quantify signaling, we first defined signaling activity as reporter fluorescence (mCitrine) normalized by receptor expression, as read out by mTurq2 fluorescence (Figure 1A, Figure 1-figure supplement 5).To control for variation in ligand expression across sender populations (Figure 1C), we further normalized this signaling activity by ligand expression, read out by a distinct cotranslational fluorescent protein (mCherry) (Figure 2-figure supplement 1A), similar to an approach used previously (Tveriakhina et al., 2018).The resulting receptor-and ligand-normalized signaling strengths varied widely across the 16 Notch-ligand-Fringe combinations (Figure 2A, Figure 2-figure supplement 1B). Comparing signaling between the two receptors revealed two key features of trans-signaling: First, almost all ligands signaled more strongly to Notch2 than to Notch1, regardless of Fringe expression (Figure 2A).The exception was Dll4, which signaled more strongly to Notch1 than Notch2 in the dLfng condition, but activated Notch1 and Notch2 to similar levels with Lfng.Second, Notch1 and Notch2 responded in a qualitatively similar way to Lfng, as can be seen by plotting signaling strengths for the Lfng condition vs. those for dLfng (Figure 2B andC).Lfng significantly enhanced Notch1 and Notch2 trans-activation by both Dll1 and Dll4.Its greatest effect was on Dll1-Notch1 (3-fold increase) followed by Dll4-Notch2 (2.5-fold increase).Lfng significantly decreased trans-activation of both receptors by Jag1 (>2.5-fold) and, to a lesser extent, Jag2 (~1.4-fold).(Note that while Jag1-Notch1 signaling was low, it was still possible to detect further reductions (Figure 2C).)Thus, the two receptors differed in their responses to the four ligands but responded similarly to Lfng, which strengthened Delta-mediated trans-activation and weakened Jagged-mediated trans-activation for both receptors. Next, we focused on the difference in trans-activation by Dll1 and Dll4 in the two Fringe conditions.In the absence of Fringe expression (i.e. with dLfng), Dll1-Notch1 signaling strength was only slightly above background (Figure 2A).By contrast, Dll4 activated Notch1 9-fold more strongly than Dll1 in the dLfng condition, consistent with previous results from E14TG2a mouse embryonic stem cells (Tveriakhina et al., 2018), possibly reflecting the ~10-fold greater binding affinity of Notch1 to the Dll4 extracellular domain (ECD), compared to the Dll1 ECD (Andrawes et al., 2013).Lfng expression increased Dll1 signaling more than Dll4, reducing the difference in signaling strengths between the ligands to 2.7-fold (Figure 2A).With Notch2, Dll1 signaling exceeded Dll4 signaling, with or without Lfng. Similar to the Delta ligands, the Jagged ligands also showed diverse signaling activities.Strikingly, Jag1 activated Notch1 poorly in both the dLfng and Lfng conditions (Figure 2A).This lack of signaling was not due to a defect in the ligand, which was properly trafficked to the cell surface (Figure 2figure supplement 2) and which signaled to Notch2 at levels comparable to those of Dll1 in the dLfng condition (Figure 2A). In contrast to Jag1, Jag2 was the strongest trans-activating ligand for both receptors with dLfng, and remained among the strongest with Lfng.Taken together, these results establish unique patterns of activity for the four ligands across different receptor and Fringe contexts. In addition to signaling strength, another key feature of signaling is ultrasensitivity, which plays a pivotal role in developmental patterning circuits (Gozlan and Sprinzak, 2023;Sprinzak et al., 2011;Yasugi and Sato, 2022).Ultrasensitivity could in principle emerge from clustering of Notch ligands and receptors (Bardot et al., 2005;Cattoni et al., 2015;Duke and Graham, 2009;Gopalakrishnan et al., 2005;Narui and Salaita, 2013;Radhakrishnan et al., 2012;Tetzlaff et al., 2018).To quantify ultrasensitivity, we titrated ligand expression in 4-epi-Tc-inducible sender cells (Figure 1-figure supplement 3A) and measured signaling activity in a cocultured minority of CHO-K1 receiver cells (Figure 2D).Signaling levels increased monotonically to levels that varied by ~1.5-fold across most ligands (Figure 2E).However, there were two exceptions: First, Dll1-Notch signaling was biphasic, declining at high trans-ligand expression levels (Figure 2D).Second, Jag1-Notch1 signaling was much weaker than other signaling interactions, as expected, preventing analysis of ultrasensitivity in this case.Most dose-response curves showed ultrasensitive responses in which signaling activity increased approximately as the square of the ligand concentration (Figure 2F, Figure 2-figure supplement 3).The exception was Jag1-Notch2 signaling, with a logarithmic sensitivity of ~1.5.An independent analysis based on fitting Hill functions to the subset of dose-response curves that reached saturation similarly produced Hill coefficients of ~2 for Dll4 with both receptors and Jag1-Notch2 (Figure 2G).The dose-response curves also allowed analysis of the relative activation strength of different ligands, defined by the ligand concentration required to signal above a threshold (Methods).Signaling strengths determined this way interpolated between the values described above in Lfng and dLfng conditions (Figure 2I), likely reflecting the endogenous Fringe profile of CHO-K1 cells, which express Rfng as well as low levels of Lfng, a profile whose effects are largely consistent with low levels of Lfng (Figure 2-figure supplement 4, Methods). Together, these results show that Notch signaling is modestly ultrasensitive for most ligandreceptor pairs in this cell context and, more broadly, reveal quantitative receptor-ligand preferences and their dependence on Lfng expression.Plated Jag1 ligands activate Notch1 more efficiently than expressed Jag1 ligands Plate-bound recombinant ligands provide a convenient method to assay Notch signaling (Kakuda et al., 2020), but differ from cell-expressed ligands in their Notch activation mechanism.We therefore sought to determine whether the method of ligand presentation (plate-bound or expressed by cells) affects ligands' signaling properties, such as signaling strength and ultrasensitivity.We analyzed CHO-K1 receiver cells' responses to titrated concentrations of plated recombinant ligand ECD, tagged at the C-terminus by a human Fc domain ('ligand-ext-Fc'), in a plated ligand assay (Figure 3A andB).As with the expressed ligand dose-response analysis, receiver cells expressed endogenous CHO-K1 Fringes. For all ligand-receptor combinations, signaling increased monotonically with plated ligand levels across the utilized concentration range.Because factors such as partial denaturation of recombinant proteins preclude absolute measurements of effective ligand concentrations, we compared the relative activity of individual ligands across different receptors (Figure 3C), but did not compare activation of the same receptor by different ligands.We again defined the relative activation strengths by the ligand concentration required to signal above a threshold (Methods).Receptor preferences were qualitatively, and sometimes quantitatively, consistent between the plated ligand assay and transactivation coculture assay (Figure 3D).For example, Jag2 and Dll1 exhibited ~2-and ~3-fold stronger activation, respectively, for Notch2 than Notch1 in both assays.Similarly, Dll4 showed a slight preference for Notch1 over Notch2 in both assays, although the fold difference was larger in the plated ligand assay.Strikingly, while Jag1 sender cells failed to activate Notch1 receivers above background in senders used for each coculture sample data point.Y-axis signaling activity values are the mean of the distribution in mCitrine (reporter activity, A.U.) divided by mTurq2 (cotranslational receptor expression, A.U.).Solid lines are fits of the increasing phase of each dataset to activating Hill functions (Methods).Dotted gray horizontal lines represent half-maximal signaling activity for each receptor across all ligand inputs.(E) Mean saturating signaling activities for indicated ligand-receptor combination, estimated by bootstrapping the Hill fits in (D), and normalized by the response to Jag2 for the same receptor.Although the decreasing phases of the Dll1 curves were excluded, saturating activity estimates may be influenced by incomplete saturation and biphasic behavior.(F) Mean logarithmic sensitivities of signaling response to ligand expression from bootstrapped linear regressions to log-log transformed, sub-saturating data points (see also This suggests that the natural endocytic activation mechanism, or potential differences in tertiary structure between the expressed and recombinant Jag1 ECD, could play roles in preventing Jag1-Notch1 signaling in coculture.Finally, in contrast to the ultrasensitive responses to expressed ligands, dose-response curves in the plated ligand assay were approximately linear for all ligand-receptor combinations except Jag1, which showed logarithmic sensitivities closer to ~1.5 for both receptors (Figure 3E, Figure 3-figure supplement 1).These differences in ultrasensitivity may reflect a difference in clustering behaviors between plated ligand-ext-Fc ligands and cell-expressed ligands (Cattoni et al., 2015).Taken together, these results indicate that plate-bound ligands differ in their Notch activation compared to ligands expressed on living cells. ", "section_name": "Trans-activation strength depends on ligand and receptor identity, and is modulated by Lfng", "section_num": null }, { "section_content": "Despite their different signaling properties, recombinant ligands provide a convenient system to measure receptor-ligand binding interactions.Previous work has shown that the effects of Lfng on binding and signaling are not directly correlated.Lfng increased recombinant Jag1 binding to Notch1, but reduced its signaling (Hicks et al., 2000;Kakuda and Haltiwanger, 2017;Taylor et al., 2014;Yang et al., 2005).On the other hand, Lfng strengthened Dll1-Notch1 binding and increased signaling.In another study, Lfng reduced Jag1-Notch2 binding (Shimizu et al., 2001).However, Lfng effects on the binding strength of most receptor-ligand combinations remain unknown. Here, we focused on Lfng effects on relative ligand-receptor binding strengths for the four Notch1ligand combinations.We incubated CHO-K1 receiver cells, or parental reporter cells expressing only endogenous receptors, with soluble ligand-ext-Fc fragments pre-clustered with a dye-conjugated antibody, as previously described (Kakuda and Haltiwanger, 2017;Varshney and Stanley, 2017; Figure 3F).We then used flow cytometry to measure the amount of ligand bound to cells, transiently expressing either Lfng or dLfng. In agreement with the previous work, Lfng strengthened Notch1 binding to Dll1 and Jag1 by 6.4-fold and 2.8-fold on average, respectively (Figure 3G and H).Lfng also modestly strengthened Notch1 binding to Jag2.However, Lfng had relatively little effect on Dll4-Notch1 binding.These data do not rule out the possibility that other ligand concentration regimes might be more sensitive to Lfng. represent the signaling thresholds defined in Figure 2D.(C) Mean signaling strengths, based on bootstrap analysis of the responses in (D) (Methods).Each signaling strength is defined as the inverse of the ligand concentration sufficient to reach threshold activity level (dotted lines in (B)), normalized to show receptors' relative activities.Colors and labels indicate ligand identity, as in Figure 2. X-axis labels are receivers; 'N' = 'Notch.'Here and in subsequent panels, error bars denote bootstrap 95% confidence intervals (Methods).(D) Comparison of mean Notch2/Notch1 signaling strength ratios with canonical trans-activation in sender-receiver cell cocultures (y-axis, values from Figure 2H) vs. the plated ligand assay (x-axis, values from (C)).(E) Mean logarithmic sensitivities computed from the slope of linear regressions to 10,000 bootstrap replicates of log-log sub-saturating signaling activities vs. ligand concentrations in Figure 3-figure supplement 1. (F) Soluble ligand binding assay (schematic, see also Methods), which enables the quantification of the strength of receptor binding to ligand-ext-Fc pre-clustered with secondary antibody.Blue in the cell nucleus represents H2B-mTurq2 fluorescence (readout of receptor expression).Star-dot-and-arrow icon refers to this specific assay.(G) Scatterplot of averaged single-cell data from the soluble ligand binding assay with Lfng (y-axis) vs. dLfng (x-axis) expression (Methods).Fluorescence background, determined as ligand bound to parental reporter cells with no ectopic Notch receptors, was subtracted, and negative values were set to zero.Solid lines are least-squares best fits, and the black dashed line is y=x.(H) Mean fold difference in the amount of each ligand bound to Notch1 with Lfng vs. dLfng from slopes of linear regressions in (G), based on bootstrap analysis of n=4 biological replicates per ligand.X-axis labels are recombinant ligands.(I) Normalized Notch signaling strength in CHO-K1 Notch1 receivers expressing Lfng or dLfng, plated on Dll1-ext-Fc (yellow) or Jag1-ext-Fc (purple) in a plated ligand assay (Methods).X-and y-axis values represent mean signaling activity (reporter activity, mCitrine, divided by cotranslational receptor expression, mTurq2), background subtracted and normalized to the maximum signaling activity-the average signal in the same receiver (+Lfng) plated on a high concentration of Dll4-ext-Fc.Solid lines are the least-squares best fits to six data points, representing three biological replicates for each of two plated ligand concentrations.Both slopes were significantly greater than or less than 1 according to a one-sided Wilcoxon signed-rank test (p-value = 0.015 for both lines).The black dashed line is y=x.Bracketed numbers are bootstrap 95% confidence intervals.(J) Cell schematic depicting the effects of Lfng expression on Jag1 and Dll1 interactions with the Notch1 receptor.White triangles represent glycosylation modifications added by Lfng, and yellow saturation level in cell nuclei represents signaling activity. The online version of this article includes the following figure supplement(s) for figure 3: In parallel with binding, we also analyzed the effect of Lfng on signaling by plate-bound ligands (Figure 3A).We incubated dLfng-or Lfng-transfected Notch1 receivers on plated Dll1-ext-Fc and Jag1-ext-Fc.Lfng strongly increased Dll1-Notch1 signaling and weakened Jag1-Notch1 signaling in the plated ligand assay (Figure 3I), similar to results with expressed ligands (Figure 2B andC).Thus, Lfng had opposite effects on binding and activation of Notch1 by the same Jag1-ext-Fc fragment.By strengthening Jag1-Notch1 binding while decreasing activation, Lfng may allow Jag1 to competitively trans-inhibit Notch1 activation by other ligands (Figure 3J; Benedito et al., 2009;Golson et al., 2009;Pedrosa et al., 2015). While they do not rule out additional effects of Lfng on binding affinity for Dll4-Notch1, these results together suggest that with Dll1, Lfng strengthens both binding and activation of Notch1, while with Jagged ligands it strengthens binding but instead weakens activation. ", "section_name": "Ligand binding is sensitive to Fringe expression", "section_num": null }, { "section_content": "Activating ligands are frequently coexpressed with Notch1 and/or Notch2, provoking the question of how ligands and receptors interact in the same cell (in cis), and more specifically whether they activate (cis-activation) or inhibit (cis-inhibition) signaling.Cis-interactions are difficult to investigate in vivo (Henrique and Schweisguth, 2019).However, in vitro studies in mammalian cells have demonstrated that Dll1 and Dll4 can cis-activate both Notch1 and Notch2 in CHO-K1 cells (Nandagopal et al., 2019), and that Dll1, Dll4, and Jag1 can cis-inhibit Notch1 activation by other ligands (LeBon et al., 2014;Preuße et al., 2015;Sprinzak et al., 2010;Thambyrajah et al., 2024).Nevertheless, it has remained unclear whether other receptor-ligand combinations also engage in cis-activation and/or cis-inhibition.We therefore sought to analyze cis-interactions more comprehensively. In the cis-activation assay (Figure 1D), we first preinduced expression of cis-ligands in receivers for 48 hr by titrating 4-epi-Tc to the desired level in the presence of the γ-secretase inhibitor DAPT, as described previously (Nandagopal et al., 2019;Figure 1E, Methods).To focus on cis-interactions alone, we then cultured receiver cells at low density, amid an excess of wt CHO-K1 cells, and allowed them to signal for 22-24 hr before flow cytometry analysis (Figure 1E andF).At these cell densities, trans-interactions among the minority cells should be minimal, activating to no more than 5% of maximum reporter activity (Figure 4A).As described below, trans-interactions between sister cells following cell division during the assay could in principle contribute to the observed cis-activation signal, but are insufficient to account for strong cis-activation. We found that cis-activation was both ligand and receptor specific (Figure 4B, left column).Notch1 exhibited no cis-activation for any of the ligands, except for a modest response to Dll4, of no more than 20% of maximal trans-activation (Figure 4B, upper left), consistent with previous observations in wt CHO-K1 cells (Nandagopal et al., 2019).By contrast, Notch2 was cis-activated by both Dll1 and Dll4, to levels exceeding those produced by trans-activation by high-Dll1 senders (Figure 4B, lower left, compare with trans-activation in Figure 4B, lower right).(Thus, the Notch2 cis-activation observed here is too strong to be explained by potential trans-interactions between sister cells following cell division in the cis-activation assay.)Jagged ligands, on the other hand, did not cis-activate Notch2 in this assay (Figure 4B, lower left). In contrast to cis-activation, the reciprocal phenomenon of cis-inhibition can only be detected in the context of basal Notch activation by other ligands.We therefore established a parallel 'cis-modulation' assay, which probes the combined effect of cis-and trans-interactions (Figure 1D, Methods).In this assay, a minority of preinduced receiver cells are cocultured with an excess of 'high-Dll1' sender cells that constitutively express Dll1 at levels sufficient to strongly activate receiver cells not expressing cisligands (Figure 4-figure supplement 1). Cis-inhibition, like cis-activation, was found to depend on both receptor and cis-ligand identity.All ligands cis-inhibited intercellular Dll1-Notch1 signaling, achieving 75-100% inhibition in the highest cis-ligand expression bin (Figure 4B, upper right).By contrast, intercellular Dll1-Notch2 signaling was cis-inhibited by Jag1 and Jag2, but not by Dll1 or Dll4, whose cis-activation further increased Notch2 signaling (Figure 4B, lower right).These results were not due to non-specific effects of ectopic cis protein expression, as Notch1 and Notch2 reporter activities showed no dependence on expression of negative control proteins H2B-mCherry or NGFR-T2A-H2B-mCherry, in either the cis-activation or cis-modulation assay (Figure 4B).Together, these results reveal three striking differences in cis-interactions among receptor-ligand combinations: (1) Delta ligands cis-activate Notch2 much more strongly than Notch1, (2) all four ligands cis-inhibit Notch1, and (3) Jagged, but not Delta, ligands cis-inhibit Notch2. ", "section_name": "Cis-interaction outcomes depend on receptor and ligand identity", "section_num": null }, { "section_content": "Since cis-activation in Delta-Notch2 receivers appeared to rival the strength of trans-activation (Figure 4B) and is blocked by γ-secretase inhibitors, similar to canonical trans-activation (Nandagopal et al., 2019), we considered the possibility that the cis-activation signal could be an artifact of intercellular signaling occurring prior to the start of the assay.During the ligand preinduction phase, the γ-secretase inhibitor DAPT prevents S3, but not S2, receptor cleavage.Thus, intercellular contacts between receivers during preinduction culture could in principle generate an S2-cleaved receptor, also known as the Notch extracellular truncation (NEXT).NEXT could then undergo S3 cleavage after DAPT removal, contributing to the total observed signal in the cis-activation assay (Figure 4C). To test this possibility, we carried out a control cis-activation assay in which receivers were cultured at both sparse and confluent densities during the 48 hr cis-ligand preinduction phase, and then replated sparsely or densely after 24 hr of preinduction to maintain the initial low or high cell density conditions (Methods).If cis-activation signal resulted from intercellular signaling during preinduction, only cells preinduced in confluent culture (able to make intercellular contacts) would show a 'cisactivation' signal 24 hr after DAPT removal.For Notch2-Dll1 and Notch2-Dll4, single-cell reporter activities correlated with cis-ligand expression, regardless of whether cells were preinduced at a high or low culture density (Figure 4D).While cells pre-cultured in the confluent condition shifted the signaling distributions to higher values relative to cells in sparse pre-cultures, the correlation between cis-ligand expression and reporter activity was maintained.This signaling shift is consistent with elevated expression of Tet-OFF-controlled cis-ligands at high cell density (Figure 4-figure supplement 2).Together, this analysis rules out the possibility that Delta-Notch2 cis-activation signal arises from S3-cleavage of NEXT generated by intercellular signaling during the ligand preinduction phase. ", "section_name": "Cis-activation does not arise from prior trans-activation", "section_num": null }, { "section_content": "In a population of cells coexpressing both receptors and ligands, signaling could occur through cisactivation, trans-activation, or both.How do these two modes of signaling combine when both can occur simultaneously?To disentangle the contributions of these two modes of signaling, we compared cis-activation alone (sparsely cultured receivers, 'cis-activation assay') to signaling occurring through both cis-and trans-interactions (confluent receivers, cis-+trans-activation assay') (Figure 1D).We repeated this analysis for each receptor-ligand pair, and for both dLfng and Lfng conditions, across a broad range of ligand expression (Figure 5-figure supplement 1). For ligand-Notch1 interactions (cis+trans), trans-activation significantly contributed to the overall signaling strength observed in our experiments (Figure 5A, Figure 5-figure supplement 2).With Lfng, the combination of cis-and trans-interactions produced over twice the signaling activity from each receptor, y-axis signaling activity was min-max normalized using the trans-signaling response to high-Dll1 senders in the cis-modulation assay (Methods, Supplementary file 3).Lines are interpolated through the mean of three biological replicates (individual data points).(C) Experimental workflow to assess the contribution of intercellular signaling during the 48 hr preinduction phase (from -72 to -24 hr) to the overall signal measured in the cis-activation assay at 0 hr (schematic).Cells were preinduced and cultured either sparsely or densely before setting up a cis-activation assay (Methods).NEXT denotes the Notch extracellular truncation, generated during the preinduction phase if cell density enables intercellular ligandreceptor interactions.Red and blue colors in cell nuclei represent H2B-mCherry and H2B-mTurq2, cotranslational reporters of cis-ligand (black) and receptor (gray) expression, respectively.Gamma-secretase is represented by the scissors.(D) 2D fluorescence distributions of signaling activity (mCitrine/ mTurq2) vs. cis-ligand expression (mCherry) in single-cell fluorescence distributions measured by flow cytometry according to the experiment in (C) performed with Notch2-Dll1 and -Dll4 receiver cells.c denotes the interval between successive cell density contours, and cell density in each discrete contour interval is indicated by color.The density interval below c is not shown.Pearson's correlation coefficient r is shown in the top left of each plot, and all p-values were <<0.001. The online version of this article includes the following figure supplement(s) for figure 4: Dll1 or Dll4 compared to cis-interactions alone in the increasing phase of the dose-response curves (Figure 5B).This result indicates that trans-activation can increase Notch1 activity beyond the level produced by cis-activation alone.For Jag2-Notch1, in either dLfng or Lfng conditions, signaling was only observed with trans-interactions (Figure 5A andB).For Jag1-Notch1, no activation was seen in the cis-activation or the cis-+trans-activation modes, consistent with earlier results (Figure 2A, Figure 4B).Ligand-Notch2 interactions showed a qualitatively different profile of behaviors compared to Notch1.Of the four ligands, trans Jag2 modestly increased Notch2 signaling beyond the level achieved by cis-activation alone, by about 1.5-fold (Figure 5B).In all other ligand-Notch2 conditions, trans-ligands failed to increase signaling, suggesting that cis-activation alone is sufficient to explain the observed level of signaling in these cis+trans conditions.With Jag1-Notch2, combined cis-and trans-signaling was slightly lower than cis-only signaling with Lfng (Figure 5B), suggesting that transinteractions may limit signaling from cis-activation in this case.(Note that this assay used a Jag1-Notch2 clone with lower background signaling than in Figure 4B, enabling detection of cis-activation (Figure 4-figure supplement 1; Figure 5-figure supplement 4).) Together, these results indicate that cis-and trans-signaling combine differently across the ligandreceptor combinations.Some combinations showed increased activity with cis-and trans-interactions compared to cis-interactions alone, while other combinations showed either a preference for a particular signaling mode or appeared to signal equally well through both modes. ", "section_name": "Coexpression of ligands and receptors can produce cis-or transsignaling in confluent monoculture", "section_num": null }, { "section_content": "Cis-activation and trans-activation share several features.They both require receptor-ligand binding at the cell surface and depend on γ-secretase cleavage (Nandagopal et al., 2019).Additionally, they share similar ligand-dependent responses to Rfng, which increases both cis-activation and transactivation by Dll1 but not Dll4 (Kakuda et al., 2020;Nandagopal et al., 2019).However, it was not clear whether Lfng would exhibit similar effects on cis-activation and trans-activation, and how any similarities or differences between activation modes might vary across receptor-ligand combinations. To address these questions, we analyzed the results from the cis-activation assay with or without Lfng (Figure 5A).Effects of Lfng on Notch1 cis-activation (Figure 5C, Figure 5-figure supplement 5) quantitatively resembled those on trans-activation (Figure 5D).Lfng significantly increased Dll1-Notch1 and Dll4-Notch1 signaling by 3.3-fold and 1.8-fold, respectively, in both assays.By contrast, Lfng had no effect on cis-activation of Notch1 by Jag1 and Jag2, which remained negligible with or without Lfng. With Notch2, Lfng's effects on cis-and trans-interactions diverged (Figure 5D).Despite its ability to increase Dll1 and Dll4 trans-activation of Notch2, Lfng did not strongly affect cis-activation.While Lfng sharply reduced Jag1-Notch2 trans-activation (Figure 2C), it had negligible effects on cis-activation by the same components (Figure 5C).Conversely, for Jag2-Notch2, Lfng had weak effects on transactivation, but reduced cis-activation by more than 2-fold (Figure 5D).Taken together, these results suggest that cis-and trans-activation mechanisms respond to Lfng similarly for Notch1, but differently for Notch2. ", "section_name": "Lfng modulates cis-activation strengths", "section_num": null }, { "section_content": "Notch trans-activation and cis-inhibition involve oligomeric binding and clustering (Chen et al., 2023;Narui and Salaita, 2013), provoking the question of whether higher order (beyond pairwise) signaling interactions could influence signaling.For example, the identity of a trans-ligand could in principle impact the effective strength of cis-interactions between a receptor and cis-ligand.To examine this The online version of this article includes the following figure supplement(s) for figure 5: Figure 5 continued possibility, we used the cis-modulation assay (Figure 1D) to determine how cis-inhibition strength depends on the identity of the trans-activating ligand for each cis-ligand-receptor pair.We cocultured each of the eight cis-ligand containing receiver cell lines (expressing endogenous CHO-K1 Fringes and preinduced to a broad range of cis-ligand levels) with an excess of each of four constitutive sender cell lines (Figure 6-figure supplement 1A and B), for a total of 32 coculture combinations (Figure 6A). The effects of cis-interactions were qualitatively similar across different trans-activating ligands (Figure 6A).In all cases, cis-Jagged ligands inhibited Notch1 and Notch2, while cis-Delta ligands inhibited Notch1 and activated Notch2 (Figures 6A and4B).(Note that cis-inhibition of Notch1 could not be analyzed when Jag1 sender cells were used due to a lack of trans-activation by Jag1 (Figure 6-figure supplement 1C).) Quantitatively, cis-inhibition strength depended on both cis-ligand and trans-ligand identity.For example, Jag2 was the most effective Notch1 cis-inhibitor across all trans-ligands (Figure 6B).Ligands' relative cis-inhibition strengths did not correlate well with trans-activation strengths (Figure 6C).In general, combinations with modest or strong cis-activating potentials, such as Dll4-Notch1, Dll1-Notch2, and Dll4-Notch2 (Figure 5A), showed relatively poor cis-inhibition efficiencies relative to trans-activation (Figure 6C).The converse was also true for Notch1: the non-cis-activating ligands, Jag1 and Jag2 (Figure 5A), showed strong cis-inhibition efficiencies relative to trans-activation (Figure 6C).For Notch2, no ligands favored cis-inhibition over trans-activation, consistent with the observation that all ligands can cis-activate Notch2 to some extent (Figure 5A). To quantitatively compare cis-inhibition efficiencies for different trans-ligands, we computed trans-adjusted cis-inhibition efficiencies by dividing values from Figure 6B by the strength of transactivation mediated by each sender population (Figure 6-figure supplement 1C).For Notch1, ligands' trans-adjusted cis-inhibition efficiencies showed diverse dependencies on the identity of the trans-ligand (Figure 6D).Notably, each cis-ligand inhibited signaling from the trans-ligand of the same identity with an equal or greater efficiency than it inhibited other trans-ligands.For example, Dll1 cis-inhibited trans-Dll1 signaling more efficiently than either trans-Jag2 or -Dll4.Similarly, Jag2 cis-inhibited trans-Jag2 signaling more efficiently than trans-Dll1 signaling, and Dll4 cis-inhibited trans-Dll4 signaling more efficiently than trans-Jag2 signaling.In contrast to the variability of Notch1 cis-inhibition, Jag-Notch2 cis-inhibition was relatively uniform across different cis-and trans-ligand combinations, varying by no more than 2-fold between the strongest and weakest cis-inhibiting combinations.Together, these results suggest that cis-inhibition strengths for Notch1, and to a lesser extent, Notch2, depend on the interplay of trans-ligand, cis-ligand, and receptor. Finally, we estimated the ultrasensitivity of cis-inhibition by fitting Hill functions to most of the doseresponse curves in Figure 6A.Hill coefficients ranged from ~0.75 to 1.5 (Figure 6E), below the values obtained for trans-activation by the same components (Figure 2F andG).These results suggest that cis-inhibition is a less ultrasensitive, or potentially more stoichiometric, process compared to transactivation.Taken together, these data suggest a complex interplay among ligands and receptors in cis and trans. ", "section_name": "Three-component interactions modulate cis-inhibition", "section_num": null }, { "section_content": "The studies above were conducted in CHO-K1 cells, but interactions between Notch receptors and ligands could in principle depend on cell type or context.To test this possibility, we constructed receiver and sender cell lines in an unrelated C2C12 mouse myoblast background, which is distinct from CHO-K1 cells, allows control of Notch component expression without altering cell fate or morphology, and has routinely been used to investigate the role of Notch signaling in muscle cell differentiation (Dahlqvist et al., 2003;Gioftsidi et al., 2022;Nofziger et al., 1999;Shawber et al., 1996).Because C2C12 cells express Notch receptors, ligands, and Fringes (Liang et al., 2021;Sassoli et al., 2012;Shimizu et al., 2001;Figure 7-figure supplement 1A), we first constructed a C2C12 base cell line, dubbed C2C12-Nkd, for 'Notch knockdown'.We used two rounds of CRISPR/Cas9 editing to target Notch2 and Jag1, the most abundant receptor and ligand, for deletion (Methods).The resulting clone (C2C12-Nkd) showed a loss of Notch2 protein by western blot (Figure 7-figure supplement 1B) and a loss of Jag1 mRNA by RT-PCR (Figure 7-figure supplement 1C).Additionally, it exhibited an impaired ability to upregulate Notch target genes, including Notch1 and Notch3 (Castel et al., 2013), relative to wt C2C12 (Figure 7-figure supplement 1A, right).We then engineered sender To compare C2C12-Nkd signaling responses to those of CHO-K1 cells, we repeated the transactivation assay described previously (Figure 1D andE) in the C2C12-Nkd background.In these assays, to minimize residual expression of endogenous Notch components, we also inserted a transient knockdown step, in which we used siRNAs to suppress basal expression of endogenous Notch receptors, and, in some cases, Rfng (Figure 7-figure supplement 1D, Methods).We focused on one ligand of each Delta and Jagged class, Dll1 and Jag1, to test whether these ligands would exhibit similar relative abilities to trans-activate Notch1 and Notch2 in the C2C12-Nkd background vs. the CHO-K1 cells. Jag1 senders activated Notch1 C2C12-Nkd receivers poorly, despite activating Notch2 receivers well (Figure 7A), just as we observed in CHO-K1 (Figure 2A).Dll1 senders activated Notch1 much more efficiently than Jag1 senders did, but Notch2 receivers were activated to a similar extent by Jag1 and Dll1 senders (<1.5-fold difference), also consistent with our results in CHO-K1. Next, to compare cis-interactions between cell lines, we performed cis-activation and cismodulation assays (Figure 1D) in the C2C12-Nkd background, adjusting assay parameters such as cell density to compensate for differences in cell properties (Figure 7B, Methods).We focused on Dll4 and Jag2, for one ligand each of the Delta and Jagged classes.With Notch2, we observed strong cis-activation by Dll4 and cis-inhibition by Jag2 (Figure 7C), similar to results in the CHO-K1 background (Figure 7D).Also similar to their behavior in CHO-K1 cells, Dll4 and Jag2 cis-inhibited Notch1 reporter activity in C2C12-Nkd cells relative to the NGFR cis-ligand negative control.Furthermore, Jag2 did not cis-activate Notch1 in this background.However, in contrast to CHO-K1, where Dll4 weakly cis-activated Notch1 even without Fringe expression (Figure 5A), we observed no cisactivation by Dll4 in C2C12-Nkd. Together, these results indicate that key signaling behaviors are similar between the CHO-K1 and C2C12 backgrounds.In particular, weak trans-activation of Notch1 by Jag1, and strong cis-activation of Notch2 by Dll1 and Dll4, are likely to be intrinsic properties of these ligands and receptors.Future studies will be necessary to extend these comparisons to a broader range of cell contexts. ", "section_name": "Similar ligand-receptor signaling features occur in a distinct cell line", "section_num": null }, { "section_content": "It has been known for decades that the mammalian Notch signaling pathway contains multiple interacting ligand, receptor, and Fringe variants.However, the functional consequences of this component diversity have remained difficult to understand.This is due in part to a lack of comprehensive measurements of signaling outcomes across ligand-receptor pairs, signaling modes (cis vs. trans), and Fringe modification states.Here, systematic mapping of interactions revealed that each ligand and receptor is unique in terms of its profile of cis-and trans-activation and cis-inhibition activities, and the dependence of these activities on Lfng (Figure 8A). Canonical trans-signaling exhibited a range of signaling activities across ligand-receptor-Fringe combinations.Ligand class (Delta vs. Jagged) did not predict the signaling level with either receptor.However, it did correlate strongly with the sign of response to Lfng, which respectively increased or decreased signaling by Delta or Jagged ligands to both receptors (Figure 2C).Amid variability in trans-signaling efficiencies, there was striking uniformity in the ultrasensitivity of Notch responses, which was roughly constant across ligand-receptor-Fringe combinations (Figure 2F andG). from Figure 2H).Cis-inhibition strength was set to zero for Delta-Notch2 combinations.(D) Cis-inhibition efficiencies and confidence intervals from (B) adjusted for the strength of trans-signaling induced by the indicated sender cells (Figure 6-figure supplement 1C), and normalized such that the maximum cis-inhibition strength is equal to 1 for each cis-ligand-receptor combination.Error bars reflect uncertainty on cis-inhibition efficiency only (trans-activation error bars were not propagated).(E) Hill coefficients (n) computed from fits of data in (A) to repressive Hill functions.Hill coefficients could not be computed for Notch1-Dll4 receivers with Dll4 or Jag2 senders because of the modest cis-activation observed at intermediate cis-ligand levels for those combinations.Values are means and 95% confidence intervals from bootstrap analysis. The online version of this article includes the following figure supplement(s) for figure 6: ", "section_name": "Discussion", "section_num": null }, { "section_content": "Sender: Dll4 (cis-modulation) Figure 7. Similar ligand-receptor signaling features occur in CHO-K1 and C2C12-Nkd cells.(A) Normalized Notch signaling strength in C2C12-Nkd Notch1 (black) or Notch2 (green) receivers cultured with Jag1 (y-axis) vs. Dll1 (x-axis) senders in a trans-activation assay.Receivers were treated with negative control (unfilled markers) or mouse Rfng (filled markers) siRNAs prior to the assay.X and y-axis values are mean signaling activity (reporter activity, mCitrine, divided by cotranslational receptor expression, mTurq2), background subtracted and normalized to the average signal in Dll1 coculture with negative control siRNA treatment for each receiver.Solid lines are the least-squares best fit through all points for a given receiver (pooling control and Rfng siRNA-treated samples, see Methods).Both slopes were significantly greater or less than 1 according to one-sided Wilcoxon signed-rank tests (p-val<0.05).The black dashed line is y=x.(B) The cell density used in the C2C12-Nkd cis-activation assay prevents intercellular signaling.After siRNA treatment to knock down residual endogenous Notch components (Methods), the assay was performed similarly to the assay used for CHO-K1 cells (Figure 4A), except for use of a ", "section_name": "C2C12-Nkd density optimization", "section_num": null }, { "section_content": "Including cis-interactions revealed additional differences among ligand and receptor variants.All ligands interacted with both Notch1 and Notch2 in cis, but did so with effects that varied across ligand-receptor-Fringe combinations.Broadly, Delta ligands cis-inhibited only Notch1, and cisactivated Notch2 at levels exceeding the maximal Notch2 trans-signaling observed here, while Jagged ligands cis-inhibited both receptors (Figure 4B).Overall, the effects of cis-ligands were complex, with signaling activity often exhibiting Fringe dependence (Figure 5C), a biphasic dependence on ligand level (Figure 5A), and, in some cases, sensitivity to trans-activating ligands (Figure 6D).Notably, Lfng had different impacts on cis-and trans-interactions for some ligand-receptor combinations such as Jag1-Notch2 and Jag2-Notch2 (Figure 5D), suggesting the possibility that cis-and trans-activation may involve different molecular mechanisms. Lfng-dependent trans-inhibition of Notch1 by Jag1 has been shown to play a key role in some contexts such as angiogenesis, where it inhibits Dll4-Notch1 signaling (Benedito et al., 2009;Pedrosa et al., 2015) and the embryonic pancreas, where it inhibits Dll1-Notch1 signaling (Golson et al., 2009).Our results explain how trans-inhibition could result from the combination of strong binding with weak activation, which was most pronounced for Jag1-Notch1 (Figure 2A, Figure 3B, and Figure 6C).Further, this effect was enhanced by Lfng, which increased binding strength in the ligand binding assay (Figure 3H), while decreasing signaling in both the coculture and plated ligand assays (Figures 2C and3I), consistent with previous observations (Hicks et al., 2000;Kakuda and Haltiwanger, 2017;Taylor et al., 2014;Yang et al., 2005).No other ligand exhibited this combination of strong binding and weak activation.These results suggest that Notch1 trans-inhibition may be a core function of Jag1, and could help explain the sometimes divergent behavior of Jag1 mutants compared with other ligands (Benedito et al., 2009;Chrysostomou et al., 2020).However, it is possible that positive Jag1-Notch1 signaling is also functionally important in some contexts, since weak signaling from Jag1-expressing OP9 cells prevents T-cell progenitors from differentiating into the B-cell lineage, in contrast with no-ligand controls (Lehar et al., 2005). A major question is how Notch receptors integrate information from cis-and trans-ligands.Previous work introduced a general model in which cis-and trans-ligands compete to form different 12-well plate.Here, y-axis values are min-max normalized, mean Notch signaling activities.Black bars are the mean of three biological repeats.(C) Results of cis-activation (left column) and cis-modulation (right column) assays for C2C12-Nkd Notch1 (top row) and Notch2 (bottom row) receivers coexpressing a Notch ligand or control protein.Fluorescence values were averaged differently for Notch1 vs. Notch2 based on responses to the nerve growth factor receptor (NGFR) control (see Methods).For both receptors, signaling activity defined in (A) was min-max normalized using the maximal trans-signaling in receivers cultured with the high-Dll4 senders (Dll4-2H10) used in the cis-modulation assay (Supplementary file 3).Lines connect means of three biological replicates (individual data points) in each mCherry bin.The bottom right plot is a zoomed-in view of the data for Notch2 receivers cultured with high-Dll4 senders, showing Jag2 cis-inhibition of Notch2 activation by Dll4 senders.(D) Comparison of cis-ligand effects in the cis-modulation assay for CHO-K1 ('CHO') vs. C2C12-Nkd ('C2C12') cell types.Y-axis units are normalized signaling activities as defined in Figure 4B for CHO-K1 cells and in (C) for C2C12-Nkd cells.(Note, maximal trans-signaling activities used in normalization differed greatly for CHO-K1 and C2C12-Nkd, so responses should be compared qualitatively, but not quantitatively (Methods).)Here, y-values are signaling activities corresponding to a cis-ligand expression level where x-axis cotranslational H2B-mCherry fluorescence equals 3×10 4 A.U., calculated by fitting a line between the x-axis mCherry bins flanking x=3×10 4 A.U. Y-values are the mean, and error bars are 95% confidence intervals, from bootstrap analysis. The online version of this article includes the following source data and figure supplement(s) for figure 7: ligand-receptor complexes.In this model, each complex may have a distinct binding strength and a distinct signaling efficiency (Formosa-Jordan and Ibañes, 2014;Sprinzak et al., 2011;Sprinzak et al., 2010).The competitive binding model is indirectly supported by previous observations that the same conserved domain of the Serrate ligand is required for both cis-inhibitory and trans-activating Notch interactions in Drosophila (Cordle et al., 2008), that interdomain flexibility in receptors and ligands could facilitate antiparallel binding in both cis and trans (Luca et al., 2015), and that synthetic synNotch ligands can cis-inhibit their receptors when expressed in the same cell (Morsut et al., 2016).However, it has not been tested systematically. Our results are broadly consistent with the competitive binding model (Figure 8B).For example, when strongly cis-activating ligands compete with strongly trans-activating ligands, they cause no net decrease in signaling, as observed when cis-Delta ligands interact with Notch2 (Figure 4B, Figure 6A).By contrast, when a strongly binding cis-ligand produces a weak or inactive complex, it can cis-inhibit.In general, combinations that did not cis-activate (Jagged-Notch1) showed relatively stronger cis-inhibition than combinations that cis-activated weakly (Delta-Notch1, Jag1-Notch2) (Figure 6C, Figure 5A).Cis-Jag2-Notch1 was the strongest cis-inhibiting combination overall.Because of its unique attributes of strong binding and weak cis-activation, cis-Jag2-Notch1 could operate in a 'mutual inactivation' regime in which a cis-ligand titrates the receptor levels available to potential trans-activating ligands (LeBon et al., 2014;Sprinzak et al., 2010;Xu et al., 2023).Linear titration of this type is consistent with the roughly linear logarithmic sensitivity observed in dose-response experiments for Notch1 with cis-Jag2 (Figure 6E). Our results revealed additional complexities in cis-inhibition.Existing models describe competitive inhibition of Notch signaling in terms of pairwise cis and trans receptor-ligand binding affinities (del Álamo et al., 2011;Formosa-Jordan and Ibañes, 2014;LeBon et al., 2014;Luna-Escalante et al., 2018;Sprinzak et al., 2010).However, in our data, cis-inhibition strength depended on the identity of the receptor, the activating trans-ligand, and the cis-ligand.Strikingly, for Notch1, each type of ligand had a comparative advantage in its ability to cis-inhibit trans-activation by itself compared to other ligands (Figure 6D).For example, Dll1 cis-inhibited trans-Dll1 signaling more efficiently than trans-Jag2, while Jag2 cis-inhibited trans-Jag2 signaling more efficiently than trans-Dll1.These results hint at the possibility of multi-way interactions, possibly involving oligomeric structures.Overall, our results were broadly consistent with earlier work for previously analyzed receptorligand pairs (Benedito et al., 2009;Hicks et al., 2000;Kakuda et al., 2020;Song et al., 2016;Stanley and Guidos, 2009;Tveriakhina et al., 2018).However, there were some notable discrepancies.While we observed a negative effect of Lfng on trans Jag1-Notch2 signaling (Figure 2C), consistent with one previous study (Shimizu et al., 2001), other studies showed either positive (Hicks et al., 2000) or no effect (Kakuda et al., 2020) with this combination.A second notable discrepancy was our observation that Delta-like ligands cis-activate but cannot cis-inhibit Notch2 (Figure 6A and Figure 7C).This conflicts with a report that wt Dll4 cis-inhibited Notch2 in U2OS osteosarcoma cells (Chen et al., 2023).It is possible that cell type-specific factors prevent Dll4 cis-activation of Notch2 in U2OS cells or that Dll4 cis-inhibition requires some interaction between Dll4 and the N2ICD (absent from our N2ECD-Gal4 receptors).Third, we found that Dll1 trans-activated Notch2 more efficiently than Notch1 (Figure 2A), consistent with results from a previous plated ligand assay (Kakuda et al., 2020), but conflicting with a coculture assay showing equivalent activation of both receptors by Dll1 (Tveriakhina et al., 2018). Looking ahead, a major question is how to understand and predict the behavior of complex pathway profiles involving multiple receptors, ligands, and Fringe proteins.For example, one of the most prevalent Notch component expression profiles, found in cell types within the heart, trachea, forelimb, and other tissues, exhibits high levels of Notch1 and Notch2 as well as moderate levels of all four activating ligands (Granados et al., 2022).What signaling properties does this combination of components produce?Which cell types can it signal to or receive signals from?Expanding the approach developed here to allow simultaneous analysis of multiple receptor activities could help to address these questions.On the other hand, directly measuring the signaling properties of all prevalent profiles may be infeasible with current techniques.Therefore, it will be critical to develop quantitative models that predict signaling behaviors among cells based on their pathway expression profiles.The data and analysis provided here should help to create such models, and thereby improve our ability to understand, predict, and control Notch signaling in diverse contexts. ", "section_name": "Figure 7 continued on next page", "section_num": null }, { "section_content": "", "section_name": "Methods", "section_num": null }, { "section_content": "The pEV-2xHS4-UAS-H2B-Citrine-2xHS4 reporter construct is the same base construct used in Nandagopal et al., 2019, with the modification of the 2xHS4 insulating elements flanking each side of the expression cassette.All of the Notch receptor piggyBac constructs were derived from the vector PB-CMV-MCS-EF1-Puro (System Biosciences), with changes made to the promoter (CMV changed to PGK or CAG) and selection marker (Puromycin to Neomycin), as well as insertion of the NotchECD-Gal4esn-T2A-H2B-mTurq2 sequence into the MCS.The Lfng and Lfng (D289E) sequences were cloned into the MCS of the original PB-CMV-MCS-EF1-Puro plasmid to create the Fringe constructs, while the IFP2.0 sequence was cloned into the same base vector as the L-Fringes, but with the Puro resistance gene replaced by the Neomycin resistance gene.Tet-OFF constructs were designed from the original pCW57.1-MAT2Aplasmid obtained by Addgene.The sequence from the end of the TREtight promoter to the beginning of the Blast resistance gene promoter was removed and replaced with each of the activating Notch ligand sequences fused to a T2A-H2B-mCherry sequence or with an H2B-mCherry or NGFR-T2A-H2B-mCherry sequence in the case of the control plasmids.For the constitutively expressing ligand constructs, the TRE-tight promoter was also removed and replaced with the CBh promoter.The plasmids used to PCR the gRNA for the CRISPR-Cas9-mediated knockout of endogenous Notch2 and Jagged1 in C2C12 cells (see CRISPR section below) were the same plasmids used in Nandagopal et al., 2019.Cell culture and plasmid transfections CHO-K1 cells (ATCC) were cultured in alpha MEM Earle's Salts (FUJIFILM Irvine Scientific) supplemented with 10% FBS (Avantor, VWR), and 1X Pen/Strep/L-glutamine (Thermo Fisher Scientific) as previously described (Elowitz et al., 2018).Transfection of CHO-K1 cells was performed using Polyplus-transfection jetOPTIMUS DNA Transfection Reagent (Genesee Scientific) according to the manufacturer's instructions.Briefly, for cells plated in 24 wells the night before (to reach ~80% confluency at time of transfection), 500 ng of non-piggyBac DNA was used along with 0.5 µL of transfection reagent.For piggyBac constructs, 500 ng of DNA+100 ng of the Super piggyBac transposase (System Biosciences) was used.For generation of stable cell lines, cells were incubated in 0.5 mL media with DNA+transfection reagent overnight at 37°C, 5% CO 2 before changing media the next day.For transient transfections, cells were incubated with DNA+transfection reagent for only 4-6 hr before media change. C2C12 cells (ATCC) were cultured in DMEM with high glucose, no glutamine (Thermo Fisher Scientific) supplemented with 20% FBS, 1X Pen/Strep/L-glutamine, and 1X sodium pyruvate (Thermo Fisher Scientific).Cells were split to ensure that stock cell cultures never reached more than 80-90% confluency.Plasmid transfection of C2C12 cells was performed using the same protocol as used for CHO cells mentioned above. All cell lines were originally authenticated by the manufacturer, ATCC, through STR profiling.ATCC also certified the cells to be free from mycoplasma contamination.Subsequent testing for mycoplasma contamination was also performed by our lab using the InvivoGen MycoStrip test kit and protocol.All cell lines were found to be free of mycoplasma contamination at the time of experiments. ", "section_name": "Plasmid construction", "section_num": null }, { "section_content": "Lentivirus was produced using the ViraPower Lentiviral Expression System (Thermo Fisher Scientific).Briefly, 293FT producer cells in a T-25 flask were transfected with a pCW57.1 expression construct (1.3 µg DNA) along with a packaging plasmid mix consisting of pVSV-G, pLP1, and pLP2 in a 2:1:1 ratio (3.9 µg DNA total).24 hr after transfection, cell media was changed with 4 mL of fresh media.48 hr post-transfection, virus containing cell media was collected and centrifuged at 3k rpm for 15 min at 4°C to remove cell debris and filtered through a 0.45 µm PVDF filter (EMD Millipore).200 µL unconcentrated viral supernatant was added to cells plated at 20,000/24 well the day before, in a total volume of 300 µL (100 µL media+200 µL virus) and incubated at 37°C, 5% CO 2 .24 hr post-infection, virus containing media was removed and replaced with fresh media.At 48 hr post-infection, cells were placed under selection with media containing 10 µg/mL blasticidin (Invivogen).After two-cell passages in selection media, cells with high mCherry expression were sorted (see Cell line construction section) and used to screen for clones. ", "section_name": "Lentivirus production and infection", "section_num": null }, { "section_content": "CHO-K1 cells were engineered to produce Notch receiver cells, receiver cells with inducible ligand, and Notch ligand sender cells.Receiver cells were created by initial transfection of the 2xHS4-UAS-H2B-mCitrine-2xHS4 plasmid.After selection in 400 µg/mL Zeocin (Thermo Fisher Scientific), cells were placed into limiting dilution, and a single reporter clone was identified that activated well in response to transient expression of Gal4, and continued with.The reporter clone was then transfected with a chimeric Notch1 or Notch2 receptor, whose intracellular domain was replaced with a Gal4esn-T2A-H2B-mTurq2 sequence (Gal4esn = minimal Gal4 transcription factor).Transfected reporter cells were selected in 600 µg/mL Geneticin (Thermo Fisher Scientific) and placed into limiting dilution.A Notch1 or Notch2 receiver cell clone was chosen by its ability to demonstrate high mCitrine expression when plated on plate-bound ligands in culture.For receiver cells with inducible ligand, Tet-OFF-ligand-T2A-H2B-mCherry was added to the receiver cells by lentiviral infection (see Lentiviral production and infection section above).Cells were selected in 10 µg/mL blasticidin (Invivogen), and sorted (Sony MA900 Multi-Application Cell Sorter) for expression of Notch (mTurq2) and ligand (mCherry) in the absence of 4-epi-Tc.Sorted cells were placed into limiting dilution, and clones were selected that demonstrated good ligand induction range (mCherry levels) when treated with various amounts of 4-epi-Tc as well as expressed good levels of mTurq2 (Notch receptor).Sender cell lines, stably expressing each of the four ligands under control of the Tet-OFF system or constitutively activated by the CBh promoter (Figure 1A), were created by infection of CHO-K1 cells with Tet-OFFligand-T2A-H2B-mCherry lentivirus or CBh-ligand-T2A-H2B-mCherry lentivirus, respectively.After selection in 10 µg/mL blasticidin, sorting for high mCherry levels and placement in limiting dilution, cell clones were chosen by their tunability of the Tet-OFF ligand with 4-epi-Tc or by the level of ligand expression in the case of constitutively expressed ligand. C2C12 receivers, receivers with inducible ligand, and sender cell lines were constructed in the same manner as the CHO-K1 cells with one major difference.C2C12 wt cells were first depleted of endogenous Notch2 and Jagged1 by CRISPR-Cas9 knockout before any cell lines were made (see CRISPR section below). ", "section_name": "Cell line construction", "section_num": null }, { "section_content": "Endogenous Notch2 and Jagged1 genes were knocked out in C2C12 mouse myoblast cells using two rounds of transfection with RNPs (ribonucleoproteins) consisting of gRNAs targeting Notch2 and Jagged1 complexed with Cas9 protein, along with an empty plasmid containing the blasticidin resistance gene added to the transfection mixture.To make the RNPs, gRNA sequences were first placed into the pX330 CRISPR-Cas9 plasmid (Cong et al., 2013) as described in Nandagopal et al., 2019.The gRNA sequences were then PCR amplified with a forward primer containing a T7 promoter and a reverse primer containing a short pA tail: Notch2 gRNA primer sequences: (5' to 3') T7 mN2C2 F TCTA CC TAAT ACGA CTCA CTAT A GGGT GGTA CTTG TGTG CC (T7 promoter) mN2C2 R AAAA GCAC CGAC TCGG TG Jagged1 gRNA primer sequences: (5' to 3') T7 mJ1C1 F TCTA CC TAAT ACGA CTCA CTAT A GCGG GTGC ACTT GCG (T7 promoter) mN2C2 R AAAA GCAC CGAC TCGG TG Resulting PCR products were transcribed using the Megashortscript T7 transcription kit (Thermo Fisher Scientific), following the manufacturer's directions.125 ng of each gRNA was separately incubated with 250 ng of Cas9 protein (PNA Bio Inc) for 10 min at room temperature, after which, the two gRNA/Cas9 mixtures were combined and used to transfect cells along with plasmid containing blasticidin resistance.Transfected cells were incubated for 24 hr before transfection media was replaced with media containing 10 µg/mL blasticidin (Invivogen).Cells were incubated for 30 hr, after which, selection media was removed and replaced with fresh media without selection.After the selected cell population was grown to ~80% confluency, cells were placed into limiting dilution in 96-well plates, calculating for 1 cell/well.Single-cell clones were identified and grown for ~10-12 days before expanding.Screening of C2C12 clones for reduced surface expression of Notch2 and Jag1 enabled identification of clone 'Nkd' that showed a loss of Notch signaling with impaired upregulation of Notch target genes Hey1, HeyL, Notch1, Notch3, and Jag2, when cells were cultured on plated recombinant Dll1 (Dll1-ext-Fc) and analyzed by RNA-sequencing (Figure 7-figure supplement 1A).The C2C12-Nkd clone also showed loss of Notch2 protein by western blot (Figure 7-figure supplement 1B) and a loss of Jag1 mRNA by RT-PCR (Figure 7-figure supplement 1C).Western blot was performed as described in Nandagopal et al., 2019.For RT-PCR analysis, RNA from cell lines was extracted by using the RNeasy Mini Kit (QIAGEN) with the cell-lysate first being homogenized through a QIAshredder column (QIAGEN), per the manufacturer's directions.RNA was then reversetranscribed with the iScript cDNA Synthesis Kit (Bio-Rad), and PCR was carried out using the Jagged1specific primers (5' to 3'): mJ1 C1 F CCAA AGCC TCTC AACT TAGT GC mJ1 C1 R CTTA GTTT TCCC GCAC TTGT GTTT RNA-sequencing data collection and analysis of C2C12 wt and Nkd clone C2C12-Nkd or C2C12 wt cells were plated at 80,000 cells/well in a 12-well plates treated with recombinant ligand (2 µg/mL Dll1-ext-Fc in PBS) or PBS only (negative control, C2C12-Nkd only) in 10 µM DAPT (Notch signaling inhibitor) overnight in two biological replicates.The next morning, DAPT was washed out and cells were allowed to signal for 6 hr before cells were harvested for RNA extraction using QIAshredder columns (QIAGEN) and the RNeasy Mini Kit per the manufacturer's instructions (see next section for further details of the plated ligand assay).cDNA libraries were prepared according to standard Illumina protocols at the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech.SR50 sequencing (10 libraries/lane) with a sequencing depth of 20-30 million reads was performed on a HiSeq2500.TrimGalore was used to run Cutadapt to trim low-quality ends (with -q 28), to remove adapters, and to clip 3 bp from the 3' ends of reads after adapter/quality trimming, as well as to run FastQC for quality assessment.Data were then uploaded to Galaxy for subsequent processing.Reads were aligned to the Mouse Dec. 2011 (GRCm38/mm10) genome using HISAT2 with default parameters and transcript abundances were computed with StringTie using the GENCODE annotation of the mouse genome (GRCm38), version M22 (Ensembl 97), downloaded June 27, 2019. Analysis of publicly available, pre-processed CHO-K1 transcriptomic data from RNA-sequencing We computed mRNA expression levels for Notch genes in CHO-K1 cells (Supplementary file 1) using processed RNA-sequencing data from CHO-K1 cells downloaded from the CHO gene expression visualization application (CGEVA) located at https://anksi.shinyapps.io/biosciences/(Singh et al., 2018).Gene expression was given by the CGEVA application in units of normalized Log2-transformed counts per million reads mapped (CPM), with normalization procedures previously described by the authors (Singh et al., 2018).For Notch genes of interest, we averaged expression measured in two different samples from CHO-K1 wt cells, and converted Log2-transformed CPM values to CPM units by the transformation CPM = 2^(Log 2 (CPM)). ", "section_name": "CRISPR-Cas9 knockout of endogenous C2C12 Notch2 and Jagged1", "section_num": null }, { "section_content": "To optimize pre-culture conditions in experiments using cell lines with Tet-OFF inducible ligands, we analyzed the dynamics and density dependence of transcription.First, we analyzed transcription dynamics with a qRT-PCR time course.CHO-K1 senders with integrated Dll1 or Dll4 ligands driven by the Tet-OFF promoter were seeded at a density that would reach confluence in a 24-well plate at the indicated time of collection.Seeded senders were induced to express ligand by reducing 4-epi-Tc concentration in the culture medium from 500 ng/mL to 5 or 10 ng/mL for Dll4 and Dll1 senders, respectively.Media was changed every 24 hr to replenish the proper 4-epi-Tc concentration, and cells were passaged every 72 hr, where applicable.At collection time, cells were spun down and RNA was extracted, followed by cDNA synthesis and qRT-PCR analysis.Ligand mRNA expression reached steady-state levels within 24 hr of 4-epi-Tc addition (Figure 1-figure supplement 3B). Second, we analyzed the Tet-OFF promoter's dependence on cell density.25,000 CHO-K1 Dll1 or Dll4 sender cells were seeded in a 24-well, 12-well, or 6-well plate (for high, medium, and low density, respectively) and induced to maximal ligand expression by removing 4-epi-Tc from the culture medium.Cells were collected 72 hr later and RNA expression was analyzed by qRT-PCR.Ligand expression from the Tet-OFF promoter showed a modest (<2-fold) dependence on cell culture density in the absence of 4-epi-Tc (Figure 4-figure supplement 2). ", "section_name": "Characterization of Tet-OFF promoter behavior", "section_num": null }, { "section_content": "We measured surface ligand levels in several CHO-K1 sender cell lines with the Pierce Cell Surface Protein Biotinylation and Isolation Kit following the manufacturer's protocol, followed by western blot analysis of the purified surface proteins.Western blot was performed as described in Nandagopal et al., 2019.Notch ligand protein was detected with anti-FLAG tag staining, while GAPDH and NAK ATPase staining served as negative and positive controls, respectively, for detection of surface protein isolation.Dll4 surface levels were undetectable despite cotranslational expression levels similar to a Dll1 cell line with positive signal (Figure 2-figure supplement 2), as others have observed in OP9 cells (Shah et al., 2012).However, this disparity could also reflect a potential dependence of surface protein pulldown efficiency on the number of primary amines (lysines) available for biotinylation in the ECD (Dll1 has 17, while Dll4 has only 9).Similarly, Jag1's high extracellular lysine count (44) could explain, at least in part, why its surface abundance exceeded that of the other ligands.Note that although surface protein isolation successfully depleted the cytoplasmic control and enriched it for the surface protein control, it is possible that some portion of the detected Notch ligands is residual cytoplasmic protein. ", "section_name": "Surface ligand isolation and quantification", "section_num": null }, { "section_content": "Various cellular assays enabled quantitative analysis of relative signaling activity mediated by different receptor-ligand pairs expressed in cis and/or trans (see Figures 1D, 3A and F).The general workflow for all assays was as follows (see also Figure 1E): Ligand expression was induced in either sender or receiver cells by reducing the 4-epi-Tc concentration in the culture medium to the desired level (Supplementary file 2), and receivers were incubated in the Notch signaling inhibitor DAPT to prevent reporter activation during this 48 hr preinduction phase.Media was changed after 24 hr to maintain the desired 4-epi-Tc concentration and 1 or 2 µM DAPT for CHO-K1 and C2C12 receivers, respectively.For all assays with siRNA knockdown, siRNAs were applied 7-10 hr after cell seeding such that cells were 30-50% confluent at time of transfection (see section 'siRNA transfections') and siRNAs were removed after an ~16 hr incubation.Plasmid transfections (when applicable) were performed immediately after siRNA media change (24 hr before starting an assay) when cells were ~80% confluent.Per 0.5 mL of medium in a 24-well, 500 ng of total plasmid was transfected including IFP2 plasmid only or 350 ng of an IFP2 plasmid cotransfected with 150 ng of a plasmid containing wt mouse Lfng or the Lfng D289E mutant (mutated catalytic aspartate, 'dLfng').(Note that surface Notch1 levels differed by less than 25% between the dLfng-and Lfng-transfected samples (Figure 1-figure supplement 4B).)Media was changed (replacing proper 4-epi-Tc and DAPT concentrations) 4-6 hr after transfection.To start the signaling assay, receivers (and sender cells, if applicable) were trypsinized using 0.25% Trypsin without EDTA and plated at a total cell density to reach confluence after 24 hr, either in a monoculture, coculture, or on plated ligand (see assay subtype details below).Senders used for each coculture assay experiment are listed in Supplementary file 3. Cells were incubated for 22-24 hr in the proper 4-epi-Tc concentration (if applicable) but without DAPT to allow signaling, and with the IFP2 cofactor biliverdin for samples transfected with IFP2 plasmid.Cells were harvested by trypsinization and Notch activation was analyzed by flow cytometry.All assays were performed with at least three biological replicates.Procedural details specific to each assay subtype, including cell coculture ratios, are described below. ", "section_name": "Signaling assays with flow cytometry readout", "section_num": null }, { "section_content": "For assays with CHO-K1 cells, wells of a 48-well plate were coated with recombinant ligand (Bio-Techne/R&D Systems) diluted in PBS to a volume of 150 µL/well and incubated at room temperature for 1 hr with rocking.For experiments with C2C12 cells, 24-well plates were used and volumes were scaled up accordingly.Either PBS alone or IgG1 Fc was plated as a negative control.The ligand or control solution was removed, and receiver cells were trypsinized using 0.25% Trypsin without EDTA and plated at 50,000 cells/well to start the assay.Plated ligand concentrations used for each experiment are given in Supplementary file 4. ", "section_name": "Plated ligand assay", "section_num": null }, { "section_content": "Excess sender cells (stable or preinduced Tet-OFF senders) or negative control senders expressing no ligand (CHO-K1 wt or C2C12-Nkd parental cells, corresponding to the receiver cell type) were cocultured with receiver cells (trypsinized in 0.25% Trypsin without EDTA) in 48-well, 24-well, or 12-well plates with a sender:receiver cell ratio of 5:1 or greater and a total cell density equivalent to 150,000 total cells/well in a 24-well plate.When receiver clones with Tet-OFF ligands were used, cis-ligand expression was suppressed with maximal [4-epi-Tc] (500-800 ng/mL).Signaling activity was analyzed by flow cytometry after 22-24 hr of signaling. ", "section_name": "Trans-activation assay", "section_num": null }, { "section_content": "Ligand expression was induced in receiver cells as described above.150,000 cells expressing no ligand (CHO-K1 wt or C2C12-Nkd parental cells, corresponding to the receiver cell type) were cocultured with 5000 receiver cells (trypsinized in 0.25% Trypsin without EDTA) in 24-well plates or 12-well plates for CHO-K1 and C2C12-Nkd cells, respectively.Signaling activity was analyzed by flow cytometry after 22-24 hr of signaling. ", "section_name": "Cis-activation assay", "section_num": null }, { "section_content": "Ligand expression was induced in receiver cells as described above.150,000 sender cells (stable or preinduced Tet-OFF senders) or negative control senders expressing no ligand (CHO-K1 wt or C2C12-Nkd parental cells, corresponding to the receiver cell type) were cocultured with 5000 receiver cells (trypsinized in 0.25% Trypsin without EDTA) in 24-well plates or 12-well plates for CHO-K1 and C2C12-Nkd cells, respectively.Signaling activity was analyzed by flow cytometry after 22-24 hr of signaling. For Figure 6 experiments only (the analysis of cis-inhibition dependence on trans-ligand identity), a slightly different culture format was used (10,000 receiver cells with 100,000 sender cells in a 48-well plate).The sender populations selected for this assay differed in their ligand expression levels (Figure 6-figure supplement 1A and B) and trans-activation efficiencies (Figure 6-figure supplement 1C).High-Dll1 and -Dll4 senders ('Dll1-L2' and 'Dll4-L2') showed similar cotranslational mCherry fluorescence.mCherry expression in high-Jag1 ('Jag1-L1') and medium-Jag2 ('Jag2-A') senders was 25% and 60% lower than in high-Dll1 senders, respectively.In the absence of cis-ligand expression, these senders activated Notch1 and Notch2 receivers with ≤2-fold differences in overall signaling activity, except for the Jag1-Notch1 combination, which failed to signal (Figure 6-figure supplement 1C), as expected (Figure 2H). ", "section_name": "Cis-modulation assay", "section_num": null }, { "section_content": "Ligand expression was induced in CHO-K1 receiver cells as described above.150,000 receiver cells were trypsinized in 0.25% Trypsin without EDTA and plated in a 24-well plate.Receiver cells treated with siRNA knockdown and plasmid transfection were plated at a higher density of 300,000 per well to reach confluence during the course of the assay despite reduced cell viability following the transfections.Signaling activity was analyzed by flow cytometry after 22-24 hr of signaling. ", "section_name": "Cis-+trans-activation assay", "section_num": null }, { "section_content": "To determine to what extent S2 cleavage from trans-signaling during the ligand preinduction phase contributes to Notch activity measured in the cis-activation assay, CHO-K1 Notch2-Dll1 and Notch2-Dll4 receivers cells were plated either at 29k in a 96-well plate (for dense conditions) or 25,000 in a six-well plate (for sparse conditions).All cells were plated in 1 µM DAPT with varying amounts of 4-epi-Tc (Supplementary file 2).24 hr post-incubation, cells were trypsinized with 0.25% Trypsin without EDTA.Cells were then counted and replated at the same cell numbers, using the same plating conditions used to initially seed the cells.After another 24 hr of incubation, the CHO-K1 cells were trypsinized, counted, and plated along with CHO-K1 cells as described above.22 hr post-incubation, cells were run on the flow cytometer to measure Notch activation.Positive controls were set up using 5000 receiver cells (treated with 500 ng/mL 4-epi-Tc) cultured with 150,000 Dll1-L1 sender cells.All conditions were performed and run in triplicate.See also Figure 4C andD. ", "section_name": "Cis-activation assay-ruling out preinduction signaling", "section_num": null }, { "section_content": "CHO-K1 reporter cells and reporter cells with integrated Notch1 ('receiver' cells) were prepared with endogenous Fringe knockdown and Lfng or dLfng transfection (with IFP2 as a cotransfection marker) as described in the section above ('Signaling assays with flow cytometry readout').Instead of beginning a signaling assay, cells were replated on non-tissue-culture-treated polystyrene plates from Cell-Star to reduce cell attachment to the bottom of the well, enabling detachment without trypsin.After a 24 hr incubation in medium supplemented with the IFP2 cofactor biliverdin, the ligand binding assay was carried out with slight modifications from protocols described previously (Kakuda and Haltiwanger, 2017;Varshney and Stanley, 2017).Cells were detached by pipetting and spun down for 5 min at 400×g at room temperature (same parameters used for all centrifugation steps), then blocked with blocking buffer (2% BSA+100 µg/mL CaCl 2 in 1X DPBS) for 15 min.Ligands were prepared by pre-clustering ligand-ext-Fc fragments (at 2× concentrations relative to the final concentrations given in Supplementary file 4) with Alexa Fluor 594-conjugated anti-human secondary antibodies (1:1000) in blocking buffer for 1 hr in the dark at 4°C.After blocking, cells were spun down again, resuspended in 50 µL blocking buffer, and mixed with 50 µL 2× pre-clustered ligands, then incubated in the dark for 1 hr at 4°C.To evaluate Fringe effects on surface Notch levels, cells were incubated with 400 ng/mL PE-conjugated anti-Notch1 antibodies (instead of ligands) in blocking buffer at room temperature in the dark for 30 min.Cells were washed once by adding 1 mL blocking buffer before spinning down, aspirating buffer, and resuspending cells in 200 µL FACS Buffer for analysis by flow cytometry (see 'Flow cytometry analysis' section for FACS buffer composition). These receptor staining data also enabled a comparison of receptor surface levels to cotranslational expression (H2B-mTurq2 fluorescence); see Figure 1-figure supplement 1B.Although receptor antibody binding strengths could differ, it appears unlikely that higher surface levels could explain most ligands' preferential activation of Notch2 over Notch1 (Figure 2H), since Notch2 levels were lower than Notch1 levels in both surface expression and cotranslational expression (Figure 1figure supplement 1B). ", "section_name": "Soluble ligand binding assay and surface receptor quantification", "section_num": null }, { "section_content": "For CHO cells, Notch1 or Notch2 receiver cells were trypsinized using 0.25% Trypsin without EDTA (Thermo Fisher Scientific).Receiver cells were plated sparsely at 2500, 5000, or 10,000 in a 24-well plate along with an equal number of CHO-K1 'high-Dll4' (Dll4-L1) sender cells for Notch1 or 'high-Dll1' (Dll1-L1) sender cells for Notch2, and surrounded by 150,000 CHO-K1 wt cells.Cell cocultures were incubated for ~22 hr and subsequently analyzed by flow cytometry to determine Notch activation (mCitrine levels) in the receiver cells.For positive controls, 5000 receiver cells were plated with 150,000 sender cells (Dll4 with Notch1 cells, and Dll1 with Notch2 cells) in order to achieve maximal activation of the receivers (used for signal normalization).5000 receiver cells were plated with 150,000 CHO-K1 wt cells along with the gamma-secretase inhibitor DAPT (1 µM, Sigma) for use as negative controls.All experiments were performed in triplicate.See results in Figure 4A. For C2C12 cells, the assay was performed similarly to the CHO-K1 cell assay with a few key changes.C2C12 Notch1 and Notch2 receiver cells were first plated at 140,000 cells per 12-well.After ~6 hr post-plating, the cells were transfected with siRNAs targeting residual endogenous mouse N1, N2, and N3 (Figure 7-figure supplement 1D), using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific), following the manufacturer's instructions.Each siRNA was used at a final amount of 4 pmol, except for N2 siRNA which was used at 20 pmol.Cell media was changed 24 hr after siRNA transfection.48 hr post-transfection, receiver cells were cocultured in a 12-well plate at 2500, 5000, or 10,000 with an equal number of C2C12 Dll1 sender c19 cells as well as with 150,000 C2C12-Nkd cells.Positive controls were set up with 5000 receiver cells+150,000 Dll1 sender c19 cells, and negative controls consisted of 5000 receiver cells+150,000 C2C12-Nkd cells along with 2 µM DAPT.See results in Figure 7B. ", "section_name": "Density optimization for the cis-activation assay", "section_num": null }, { "section_content": "Cells were seeded in 24-well or 12-well plates at a density to reach 30-50% confluence at the time of transfection-either 7-10 hr or 24 hr after plating, depending on the experiment.For Fringe knockdown in CHO-K1 cells, 2 pmol each of siRNA targeting the endogenous Rfng and Lfng transcripts, or 4 pmol total negative control siRNA (Allstars negative control, QIAGEN), were transfected using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.For all assays with C2C12 cells, the same reagent was used to transfect cells with either negative control siRNA (32 pmol), N1+N2+N3 siRNA (4 pmol+20 pmol+4 pmol, respectively, plus 4 pmol control siRNA) or N1+N2+N3+Rfng siRNA (4 pmol+20 pmol+4 pmol+4 pmol, respectively) in a 12-well plate.C2C12-Nkd cells already exhibit reduced levels of Notch1 and Notch3 (Figure 7-figure supplement 1A); however, Notch1 and Notch3 knockdowns were performed to prevent their subsequent upregulation by signaling during the assay.The Notch2 siRNA knockdown was not essential for these experiments, but performed despite very low levels of Notch2 to begin with. In all assays, siRNAs were incubated with cells for 16-24 hr before media change, and transfected cells were harvested for knockdown quantification by qRT-PCR or for use in signaling assays 24-36 hr after initial transfection.For analysis by qRT-PCR, cells were spun down by centrifugation at 1400 rpm for 3 min at room temperature.After supernatant removal, the cell pellets were stored at -80°C for later RNA extraction using the RNeasy Mini Kit (QIAGEN) with the cell-lysate first being homogenized through a QIAshredder column (QIAGEN), per the manufacturer's directions, followed by cDNA synthesis with the iScript cDNA Synthesis Kit (Bio-Rad), and finally analyzed by qPCR using the qPCR primers in Supplementary file 5. qPCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Analysis of endogenous Fringe effects on signaling in CHO-K1 cells CHO-K1 cells endogenously express low levels of Lfng and ~20-fold higher levels of Rfng (Supplementary file 1; Singh et al., 2018).To assess the effects of endogenous Fringes on receptor-ligand interactions, we expressed dLfng or Lfng while knocking down endogenous Rfng and Lfng with siRNA in CHO-K1 receiver cells (as described above), and compared these 'No Fringe' and 'high Lfng' conditions to wt levels of Fringe in a trans-activation assay with flow cytometry readout (Figure 2-figure supplement 4). As a general trend, the magnitude of signaling in the presence of endogenous Fringes fell between the dLfng and Lfng signaling magnitudes.Jag1-Notch1 signaling was not potentiated by endogenous Fringes (as would be expected in the case of Rfng dominance), but showed 1.7-fold weaker with endogenous CHO-K1 Fringes than with dLfng, consistent with Lfng dominance (Kakuda et al., 2020;Pennarubia et al., 2021;Yang et al., 2005), despite the much lower expression of Lfng relative to Rfng (Supplementary file 1).(Note: Near signal saturating conditions, we observed very small (<1.3-fold) decreases in Delta-Notch signaling with endogenous Fringes compared with dLfng, suggesting the possibility of mild, non-specific effects of the siRNA or plasmid treatment in the two conditions.)Although endogenous Fringes showed overall weaker effects on signaling activity than transfected Lfng did, our results suggest that endogenous Lfng modestly weakens Jagged signaling and strengthens Dll1-Notch1 signaling.See also Figure 2I. ", "section_name": "siRNA transfections and qRT-PCR analysis", "section_num": null }, { "section_content": "For analysis of cells by flow cytometry, cells were trypsinized in 0.05% or 0.25% Trypsin-EDTA (Thermo Fisher Scientific).Cells were resuspended in 1X FACS buffer: 1X Hanks Balanced Salt Solution (Thermo Fisher Scientific) supplemented with 2.5 mg/mL bovine serum albumin (Sigma-Aldrich) and 200 U/mL DNAse I. Resuspended cells were filtered through 40 µm cell strainers (Corning Inc, Corning, NY, USA) into U-bottom 96-well tissue culture-treated plates.Cells were analyzed on a Beckman Coulter Life Sciences CytoFLEX benchtop flow cytometer.Data were analyzed in Python using custom software according to the following workflow: 1. Cells were gated in a 2D plane of forward scatter (FSC) and side scatter (SSC) to select intact, singlet cells.2. Cells were gated in a 2D plane of mTurq2 (PB450, A.U.) vs. SSC to separate out the +mTurq2 receiver cells from -mTurq2 senders or 'blank' parental cells (Figure 1F). 3. Plasmid-transfected cells were gated in the APC700 channel to select cells expressing the cotransfection marker IFP2 above background levels (Figure 1G).High IFP2 levels were excluded to avoid overexpression artifacts.4. Receiver cells coexpressing ligand were gated into six logarithmically spaced bins of arbitrary mCherry (ECD) fluorescence units (Figure 1B).Expression levels above the highest mCherry bin were excluded due to overexpression artifacts observed with the control proteins H2B-mCherry and NGFR. 5. Compensation was applied to subtract mTurq2 signal leaking into the FITC channel.6.If applicable, reporter activity, mCitrine (FITC, A.U.) fluorescence was normalized to cotranslational receptor expression by dividing mCitrine by the mTurq2 signal (PB450, A.U.).The resulting mCitrine/mTurq2 ratio is the 'signaling activity' (reporter activity per unit receptor).Signaling activity defined this way controls for variations in receptor expression across receiver clones and eliminates artifacts in mCitrine and mTurq2 fluorescence quantification that occur when binning cells on high cis-ligand expression (Figure 1-figure supplement 5).7. If applicable, cotranslational cis-ligand expression was normalized to cotranslational receptor expression by dividing mCherry by the mTurq2 signal (PB450, A.U.).The resulting mCherry/ mTurq2 ratio (cis-ligand expression per unit receptor) controls for slight variations in receptor expression when quantitatively comparing ligands' cis-inhibition efficiencies (Figure 6A).8. Average bulk measurements for each sample were obtained by computing the mean signal across single-cell data for a given sample (and mCherry bin, if applicable).Cells treated with different 4-epi-Tc levels were pooled as technical replicates after mCherry binning.A minimum of 100 cells were required during averaging; mCherry bins with too few cells did not generate a bulk data point.Notes: (1) Despite sometimes heterogeneous cis-ligand expression, reporter activity vs. cisligand expression curves showed similar trends in CHO-K1 cells whether fluorescence values were averaged across cells from different wells sorted into the same bin of cis-ligand (mCherry) expression (Figure 5A) or across cells from the same well and 4-epi-Tc concentration, without mCherry binning (Figure 5-figure supplement 2).(2) For C2C12-Nkd cells, the fluorescence averaging method sometimes showed differences when values were averaged across cells from different wells sorted into the same bin of cis-ligand (mCherry) expression or across cells from the same well and 4-epi-Tc concentration, without mCherry binning.We selected averaging methods based on responses to expression of the NGFR control (Figure 7C).For Notch2, individual receivers were sorted into discrete bins of mCherry (A.U.) and the mCitrine/mTurq2 (signaling activity) and mCherry (cotranslational cis-ligand expression) fluorescence values were averaged across all cells in that bin.For Notch1, single-cell data were not sorted into mCherry bins, but averaged by 4-epi-Tc concentration, because mCherry binning yielded anomalous reporter activities with the NGFR control, possibly due to selection of abnormally large cells when sorting into the high mCherry bins.9. Background subtraction was performed by subtracting 'leaky' reporter activity of the receiver (using high 4-epi-Tc concentrations to minimize cis-ligand expression, where applicable) in coculture with 'blank' senders (CHO-K1 wt or C2C12-Nkd parental cells, according to the receiver cell type).10.Y-axis normalization was performed as described in each Figure caption.The term, 'max normalized', refers to signaling activities that were divided by trans-signaling in coculture with excess senders expressing relatively high ligand levels (with cis-ligand expression suppressed by high 4-epi-Tc, where applicable.)When signaling activities were background subtracted first, they are referred to as 'min-max normalized'.Note: the maximum trans-signaling activity used in these normalizations may not reflect the true reporter activity maximum, especially in cases where cis-activation exceeds trans-activation strength.Additionally, in Figure 7D, maximal trans-signaling used in normalization was relatively weak for Notch2 (only 1.3-to 2.2-fold above background).11.In some cases, average signaling activity (from steps #6 and #8 above) was further normalized to average cotranslational ligand expression (mCherry, A.U.) to define a signaling strength metric (Figure 2A, Figure 2-figure supplement 4B).In such cases, saturated data points, defined as those with normalized signaling activity over 0.75 in both dLfng and Lfng conditions, were excluded.Only senders with mCherry levels <75% of the maximum sender expression were used, due to nonlinearities in normalized signaling activity with extremely high ligand expression.This simple normalization method reduced variance in normalized signaling activity, but does not reflect the independently observed ultrasensitivity with respect to ligand expression (Figure 2F and G). Note: all uses of 'histogram' to describe single-cell fluorescence distributions refer to kernel density estimates. ", "section_name": "Flow cytometry analysis pipeline", "section_num": null }, { "section_content": "At least three biological replicates were used for each flow cytometry and qRT-PCR experiment, where biological replicates are distinct samples prepared separately, sometimes in parallel on the same day and sometimes on different days.Two biological replicates were used for RNA-sequencing.Least squares regressions to fit data to lines or Hill functions were computed using scipy.optimize.leastsq.Activating and repressing Hill functions were defined, respectively, as y = bx n / ( K n + x n ) and y = bK n / ( K n + x n ) , where K denotes the EC50 and b denotes the curve maximum.We note that fits of activating responses to Hill functions could exhibit systematic errors in estimation of maximal signaling activity where observed responses are biphasic (e.g.Dll1 curves in Figure 2D) or do not fully saturate (e.g.Jag2 curves in Figure 2D).For the biphasic Dll1 curves, only data points in the increasing phase were fit to Hill functions. Signaling strength metrics were based on the ligand concentration at which the fitted Hill function crossed a y-axis threshold (Figure 2D and Figure 3B).These thresholds were defined as the halfmaximum signaling measured for each receptor (separately, due to differences in reporter dynamic range) from Figure 2D trans-activation data with Jag2, which signaled most strongly to both receptors.However, Jag2 curves were not fully saturated for either receptor, and errors in the estimation of relative maximum signaling for the two receptors (used to define half-maximal thresholds) could potentially skew relative signaling strengths computed for Notch1 vs. Notch2. For linear regressions to compute a fold-change between two conditions, y-intercepts were fixed to (0,0) (Figure 2B, Figure 3G , Figure 3I, Figure 5-figure supplement 3, Figure 5-figure supplement 5, Figure 7A).For statistically robust comparison of the relative trans-signaling strengths of Dll1 and Jag1 in C2C12-Nkd cells (Figure 7A), we pooled data from control and Rfng siRNA treatment conditions (Rfng is reported to potentiate Notch1 signaling but have no effect on Notch2 signaling for both Dll1 and Jag1; Kakuda et al., 2020). All mean values and 95% confidence intervals were computed based on 10,000 bootstrap replicates with at least n=3 biological replicates.When bootstrapping confidence intervals on parameter estimates from fitting ligand dose-response curves to lines (Figure 2 6A), bootstrapped datasets were constructed by sampling separately from biological replicates within each x-axis 'bin', defined as the mCherry fluorescence window, the plated ligand concentration, or the 4-epi-Tc concentration, as relevant. p-Values were computed in two ways.To evaluate whether the correlation between two paired variables had a slope greater or less than 1, we used one-sided Wilcoxon signed-rank tests implemented in scipy-scipy.stats.wilcoxon.To test whether two sets of data points came from distributions with the same means, we used permutation testing following these steps: 1. Compute the true difference in means between the control and test datasets (of length n and m, respectively).2. Pool the two datasets and scramble the order of data points.3. Define the permutation sample by labeling the first n points from the scrambled data as 'control' data and the next m points as 'test' data.4. Compute the difference in means between the control and test distributions from the permutation replicate vs. the original dataset. 5. Repeat many times.The p-value is the fraction of permutation replicates with a difference in means greater than the true difference in means. p-Values were computed using at least 10,000 permutation replicates with one-sided differences in mean. ", "section_name": "Statistical analysis", "section_num": null } ]	[ { "section_content": "RK, LS, and MBE conceived and designed the experiments RK and LS generated the cell lines and performed the experiments.RK and LS analyzed the experimental data.RK, LS, and MBE wrote the paper.We thank Irwin Bernstein for generously sharing the recombinant Dll1-ext-Fc ligand, and Igor Antoshechkin in the Millard and Muriel Jacobs Genetics and Genomics Laboratory for assistance with RNA-sequencing.We are grateful to Xun Wang and the Rothenberg Lab for providing the NGFR construct.We would also like to thank Ellen Rothenberg, David Sprinzak, Stephen Blacklow, Sandy Nandagopal, James Linton, Martin Tran, Jan Gregrowicz, Ronghui Zhu, Felix Horns, and Jacob Parres-Gold for discussions about this work and for critical feedback on this manuscript.This work was supported by the National Institutes of Health (NIH) (grant R01 HD7335C).RK was supported by an NIH Ruth L Kirschstein NRSA predoctoral fellowship (F31 HD100185).MBE is a Howard Hughes Medical Institute Investigator.This article is subject to HHMI's Open Access to Publications policy.HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles.Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Data availability RNA sequencing data are available at NCBI via the GEO accession GSE233573.All raw and processed datasets (RNA-seq, qRT-PCR, and flow cytometry) and the custom Python scripts used to make figures and support conclusions in this paper are available at data.caltech.edu: https://doi.org/10.22002/gjjkn-wrj28. The following datasets were generated: ", "section_name": "", "section_num": "" }, { "section_content": "Supplementary files Supplementary file 1. Notch gene expression in wild-type CHO-K1 cells.These expression values in counts per million reads mapped (CPM) were obtained from the CHO gene expression visualization application (CGEVA) database of CHO RNA-sequencing (RNA-seq) data, including from wild-type CHO-K1 cells, located at https://anksi.shinyapps.io/biosciences/(Singh et al., 2018).Dll1, Dll4, Jag2, and Mfng were not available in the database.See also Methods. Supplementary file 2. 4-epi-Tc concentrations used to induce ligand expression.Where Tet-OFF inducible sender or receiver cells were used but don't appear in the table above, 4-epi-Tc was used at a concentration of 500-800 ng/mL to fully suppress expression. Supplementary file 3. Sender-receiver cell pairs used for coculture assays.Where multiple receivers are listed, they were cultured with the same senders but in separate wells (receivers were not mixed together).All experiments with CHO-K1 cells used wild-type cells as negative sending controls and experiments with C2C12-Nkd receivers used the blank C2C12-Nkd parental line as negative controls.Clone identifiers are in parentheses; polyclonal cell lines lack parentheses.See senders' ligand expression distributions in Figure 1C, Figure 1 ", "section_name": "Additional information", "section_num": null }, { "section_content": "", "section_name": "Additional information", "section_num": null }, { "section_content": "Supplementary files Supplementary file 1. Notch gene expression in wild-type CHO-K1 cells.These expression values in counts per million reads mapped (CPM) were obtained from the CHO gene expression visualization application (CGEVA) database of CHO RNA-sequencing (RNA-seq) data, including from wild-type CHO-K1 cells, located at https://anksi.shinyapps.io/biosciences/(Singh et al., 2018).Dll1, Dll4, Jag2, and Mfng were not available in the database.See also Methods. Supplementary file 2. 4-epi-Tc concentrations used to induce ligand expression.Where Tet-OFF inducible sender or receiver cells were used but don't appear in the table above, 4-epi-Tc was used at a concentration of 500-800 ng/mL to fully suppress expression. Supplementary file 3. Sender-receiver cell pairs used for coculture assays.Where multiple receivers are listed, they were cultured with the same senders but in separate wells (receivers were not mixed together).All experiments with CHO-K1 cells used wild-type cells as negative sending controls and experiments with C2C12-Nkd receivers used the blank C2C12-Nkd parental line as negative controls.Clone identifiers are in parentheses; polyclonal cell lines lack parentheses.See senders' ligand expression distributions in Figure 1C, Figure 1 ", "section_name": "Additional files", "section_num": null } ]
10.1186/s43141-021-00198-z	Evaluation of MLPA as a comprehensive molecular cytogenetic tool to detect cytogenetic markers of chronic lymphocytic leukemia in Egyptian patients	Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia. This disease is genetically heterogeneous, and approximately 85% of patients with CLL harbor chromosomal aberrations that are considered effective prognostic biomarkers. The most frequent aberrations include deletions in 13q14, followed by trisomy 12, and deletions in 11q22.3 and 17p13 (TP53). Currently, fluorescence in situ hybridization (FISH) is the most widely used molecular cytogenetic technique to detect these aberrations. However, FISH is laborious, time-consuming, expensive, and has a low throughput. In contrast, multiplex ligation-dependent probe amplification (MLPA) is a reliable, cost-effective, and relatively rapid technique that can be used as a first-line screening tool and complement with FISH analysis. This study aimed to evaluate the contributions of MLPA as a routine standalone screening platform for recurrent chromosomal aberrations in CLL in comparison to other procedures. Thirty patients with CLL were screened for the most common genomic aberrations using MLPA with SALSA MLPA probemix P038-B1 CLL and FISH.In 24 of the 30 cases (80%), the MLPA and FISH results were concordant. Discordant results were attributed to a low percentage of mosaicism. Moreover, the MLPA probemix contains probes that target other genomic areas known to be linked to CLL in addition to those targeting common recurrent CLL aberrations.The usage of MLPA as the first screening platform followed by FISH technique for only the negative cases is the most appropriate approach for CLL diagnosis and prognosis.	[ { "section_content": "Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in western countries, accounting for 30% of all leukemia cases.However, it is infrequent in the Eastern world.In Upper Egypt, CLL accounts for around 11.3% of all leukemia cases.This hematopoietic neoplasm arises from B-lymphocytes in the peripheral blood, bone marrow, and/or lymph nodes [1,2].Moreover, CLL is a genetically heterogeneous disease, and the clinical course may range from months to decades.Approximately 85% of CLL patients harbor chromosomal aberrations, which are considered effective prognostic biomarkers.The most frequent aberrations involve deletions in 13q14 (50-60%), which are associated with a good prognosis.The next most frequent aberration is trisomy 12 (12-25%), which is associated with intermediate prognosis, followed by 11q22.3(ATM; 10-20%) and 17p13 (TP53; 5-10%) deletions, which are associated with a poor prognosis.These aberrations are important prognostic biomarkers for treatment decisionmaking [3]. Currently, fluorescence in situ hybridization (FISH) is the most widespread molecular cytogenetic technique used to detect genetic abnormalities in CLL [4].However, FISH cannot detect small or intragenic deletions.Moreover, FISH is a laborious, time-consuming, expensive, and low-throughput procedure relative to other molecular genetic procedures used to detect common aberrations.Several other chromosomal aberrations in CLL have been detected using different techniques.However, these aberrations are not usually analyzed in clinical practice [5]. Multiplex ligation-dependent probe amplification (MLPA) was first introduced in 2002 [6].This multiplex PCR technique can detect abnormal copy numbers in up to 50 different genomic DNA or RNA sequences and can differentiate sequences differing in only one nucleotide.Up to 96 samples can be tested simultaneously by MPLA, and the turn-around time is within 24 h.Consequently, MLPA has considerably increased the detection rates of various genetic disorders [7].MLPA has also been applied successfully to the detection of copy number abnormalities in various malignant hematopoietic disorders, such as CLL [8].MLPA is a reliable, costeffective technique and is more rapid than FISH.Although MLPA cannot detect low-level mosaicism, it remains useful as a first-line screening tool and complement with FISH analysis [9].The commercially available SALSA MLPA probemix P038 was designed specifically for CLL screening and permits the concurrent evaluation of various risk-linked genomic targets.This kit contains probes for 10q (PTEN), 11q (ATM, RDX, PPP2R1B, CADM1), chromosome 12, 13q14 (RB1, DLEU1/2/7, KCNRG, MIR15A), 14q, 17p (TP53), and chromosome 19.This study aimed to evaluate the contributions of MLPA as a routine standalone screening platform for recurrent chromosomal aberrations in CLL in comparison to other procedures such as FISH. ", "section_name": "Background", "section_num": null }, { "section_content": "This study was conducted at the National Research Centre, Egypt, and was approved by its Medical Ethical Committee.Informed written consent was obtained from the study participants.Thirty CLL patients (16 males, 14 females) were included in this study.The average age at the time of sampling was 65 years (range, 36-88 years).All participants attended the National Cancer Institute, Cairo University, Egypt.The diagnosis of CLL was established according to the World Health Organization classification of hematolymphoid tumors [10].CLL was diagnosed by the presence of at least 5000 monoclonal B-lymphocytes/μl with a CLL immune phenotype in the peripheral blood (PB) for at least 3 months.Typically, CLL lymphocytes are small and mature-looking, with scanty cytoplasm and a dense nucleus containing partially aggregated chromatin.PB samples were collected on heparin to enable blood culture and on K2-EDTA in a vacutainer tube to allow DNA extraction. ", "section_name": "Methods", "section_num": null }, { "section_content": "Peripheral heparinized blood samples were cultured without mitogens and incubated at 37°C for 24 h.Cell harvesting and slide preparation were performed using the standard conventional cytogenetic methods. FISH analysis was performed according to the manufacturer's instructions and Pinkel et al. [11], using FISH probes for the most common genomic aberrations associated with CLL, including trisomy 12 and deletions at the 13q14, 11q22, and 17p13 loci.All FISH probes were commercially available (Cytocell, UK).The slides were examined using a suitable filter set on an optimally performing fluorescence microscope with an applied imaging system.A total of 200 interphase cells were examined per patient. ", "section_name": "FISH analysis", "section_num": null }, { "section_content": "DNA was extracted from the PB lymphocytes of all 30 cases and reference samples (one reference sample per seven patient samples, with a minimum of three references per test) using the QIAamp DNA Mini Kit (Germany) according to the manufacturer's instructions.The quality and quantity of the DNA samples were determined using a NanoDrop spectrophotometer. The MLPA assay was performed using SALSA MLPA probemix P038-B1 CLL according to the manufacturer's instructions (MRC-Holland, Netherlands).This probemix comprises multiple probes specific for chromosomal regions and genes associated with recurrent copy number aberrations in B-lymphocyte CLL, including 10q23.31(PTEN), 11q 22 (ATM, RDX, PPP2R1B, CADM1), chromosome 12, 13q14 (RB1, DLEU1/2/7, KCNRG, MIR15A), 14q, 17p (TP53), and chromosome 19.Moreover, the P038 probemix contains three probes to detect the NOTCH1 7541-7542delCT, SF3B1 K700E, and MYD88 L265P mutations, which only produce a signal when the precise mutation is present.The assay kit included SD009 sample DNA as a positive control for the mutation-specific probes and data binning in the fragment analysis. The DNA denaturation and overnight MLPA probemix hybridization steps were followed by probe ligation and amplification on the following day.The amplified products were separated using an ABI 3500 Analyzer (Applied Biosystems, USA).The results were interpreted using the Coffalyser.Net software (MRC, Holland).Ratios of <0.75, 0.75-1.30,and >1.3 were considered to indicate deletion, normal, and duplication, respectively. ", "section_name": "MLPA assay", "section_num": null }, { "section_content": "Samples from 30 patients with CLL were studied.The FISH and MLPA results are summarized in Table 1. FISH detected aberrations in 21 cases (70%), whereas no abnormalities were detected in nine cases (30%).The most common defect was trisomy 12, which was present in 12 patients (40%).A 13q14 deletion was detected in 10 cases, while an 11q22 deletion was observed in four cases, and a 17p13 deletion was detected in three cases (Table 2, Fig. 1). MLPA detected aberrations in 20 cases (66.7%) and no abnormalities in the remaining 10 cases (33.3%).The most common abnormality was trisomy 12, which was present in nine cases (30%).A 13q14 deletion was detected in nine cases, while the RB1 gene was not included in the deleted area in four cases.The 17p13 deletion and 11q22 deletion were detected in three cases each, and the 14q deletion and trisomy 19 were observed in one patient each (Table 2).NOTCH1 7541-7542delCT, SF3B1 K700E, and MYD88 L265P mutations were not detected in any of the patients (Fig. 2). ", "section_name": "Results", "section_num": null }, { "section_content": "Many authors have recommended the use of MLPA as an initial diagnostic test [12,13].In this study, we aimed to determine the usefulness of the MLPA probemix P038-B1 as a routine standalone screening platform for the detection of clinically relevant chromosome abnormalities in CLL. Samples from six of the 30 studied patients (20%) yielded discordant MLPA and FISH results.Two cases had a 14q deletion and trisomy 19 respectively, which were not evaluated by FISH in this study.Five cases harbored abnormalities that were identified by FISH but not by MLPA.So, MLPA results were consistent with the FISH results in 24 of 30 patients (80%).Fabris et al. [8] detected a 95% concordance rate between MLPA and FISH results in CLL.The discordant results in these cases may be related to a low level of mosaicism.However, the definition of low mosaicism, or the level at which abnormalities could not be detected by MLPA, has differed between studies and remains controversial.For example, the reported mosaicism thresholds have ranged from 36% in a study by Al Zaabi et al. [9] to 20% in a study by Abdool et al. [14].However, false-negative MLPA results were reported in samples with an aberrant cell percentage <25% [15,16].In our study, abnormalities could be detected by MLPA in sample containing 27% mosaicism.Interstitial at 13q14 is the most common chromosomal aberration in CLL and is detected in approximately 50% of cases.The deletion of 13q as the sole abnormality is strongly associated with a favorable disease outcome and a better prognosis.Studies suggest that the clinical course of CLL is accelerated in patients with a large 13q14 deletion that includes the RB1 gene.Moreover, reciprocal translocations involving 13q14 [t(13q)] and many different chromosomes have been reported.However, the lack of recurrent other abnormalities suggests that the consequence of these translocations is possibly due to the loss of a tumor suppressor gene rather than the generation of a fusion gene [17].In our study, five of the nine cases in which a 13q deletion was detected by MLPA were affected by large deletions that included the RB1 gene. With sufficient accumulated genotoxic damage, CLL cells are directed to undergo cell cycle arrest or apoptosis.ATM and TP53 genes govern the cellular response to DNA damage through the ATM-CHK2-p53 signaling pathway.Alterations of these genes lead to genomic instability and chemoresistance and are associated with adverse prognosis with significantly shorter overall survival [18][19][20]. 11q deletion, which causes a loss of the ATM gene at 11q22.3, is detected in 25% of CLL cases.This is the most frequently detected unfavorable genetic anomaly in patients with CLL.Larger 11q deletions also occur and may affect the tumor suppressor genes PPP2R1B, CADM, and RDX [21,22].In our study, all patients in whom MLPA detected an 11q deletion were affected by large deletions that included these tumor suppressor genes. While 17p deletion causes a loss of the tumor suppressor gene TP53 at 17p13.1 and is associated with a rapid disease progression, poor outcome, drug resistance, and reduced survival in the literature, the incidence of 17p deletion varies from 3.4 to 16.8% [23,24].In our study, both MLPA and FISH detected 17p deletions in three cases (10%).Trisomy 12 is the third most common chromosomal aberration detected in patients with CLL.This abnormality is identified in 10-20% of patients [25].In our study, MLPA and FISH detected trisomy 12 in nine and 12 cases, respectively.This discordance was attributed to the previously discussed low level of mosaicism in three cases. In addition to four probes that target common recurrent CLL aberrations, the MLPA probemix contains probes that target other genomic areas known to be linked to CLL.These areas, namely, 10q (PTEN), 14q, and chromosome 19, are not targeted by the FISH probe panel.PTEN is a tumor suppressor gene.PTEN is impaired in several types of cancers and plays an important role in CLL pathogenesis.Studies have described defective PTEN function in CLL, either through gene mutation/deletion or promoter methylation [26,27].While 14q deletions are rare recurrent alterations in CLL frequently associated with trisomy 12, 14q deletions are associated with a short time to treatment.14q deletions seem to have an adverse prognostic impact when associated with trisomy 12 [28].Also, trisomy 19 has been detected infrequently in CLL cases and is usually associated with trisomy 12 [29].In our study, no abnormalities were detected in the 10q (PTEN) region.Only one case harbored a 14q deletion, and one case harbored trisomy 19, and both cases were having associated trisomy 12. Moreover, the P038 probemix includes probes to detect three mutations: NOTCH1 7541-7542delCT, SF3B1 K700E, and MYD88 L265P.These mutations are recently identified as CLL disease parameters.The presence of these mutations is associated with at least one unfavorable prognostic marker [30][31][32][33].However, these mutations were not detected in any of our patients. ", "section_name": "Discussion", "section_num": null }, { "section_content": "From our study, both assays have comparable capabilities to detect CLL aberrations.MLPA technique is disadvantaged by its inability to detect targeted abnormalities in a sample with a low level of mosaicism.However, the restricted number of regions that can be evaluated by FISH is considered to be disadvantageous.MLPA is more cost-efficient than FISH and encompasses a broader range of target gene loci.Nevertheless, we recommend the usage of MLPA as the first screening platform followed by FISH technique for only the negative cases as the most appropriate approach for CLL diagnosis and prognosis. ", "section_name": "Conclusions", "section_num": null } ]	[ { "section_content": "We would like to express our gratitude to the National Research Centre, Egypt, for giving us the chance to accomplish this study with the help of its updated equipment and instrumentation. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "The MLPA assay part of this research was supported by In-house Research Project grant, grant number: AR111303, National Research Centre, Egypt. ", "section_name": "Funding", "section_num": null }, { "section_content": "Data and material are available upon request. ", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "The study was approved by the ethical committee of the National Research Centre (18-047), which is in accordance with the ethical standards of the Declaration of Helsinki.All participants gave informed written consent before their inclusion in the study. Not applicable. The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Declarations", "section_num": null }, { "section_content": "", "section_name": "Declarations", "section_num": null }, { "section_content": "The study was approved by the ethical committee of the National Research Centre (18-047), which is in accordance with the ethical standards of the Declaration of Helsinki.All participants gave informed written consent before their inclusion in the study. ", "section_name": "Ethics approval and consent to participate", "section_num": null }, { "section_content": "Not applicable. ", "section_name": "Consent for publication", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.3389/fimmu.2021.784691	Bone Marrow Lymphoid Niche Adaptation to Mature B Cell Neoplasms	<jats:p>B-cell non-Hodgkin lymphoma (B-NHL) evolution and treatment are complicated by a high prevalence of relapses primarily due to the ability of malignant B cells to interact with tumor-supportive lymph node (LN) and bone marrow (BM) microenvironments. In particular, progressive alterations of BM stromal cells sustain the survival, proliferation, and drug resistance of tumor B cells during diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL). The current review describes how the crosstalk between BM stromal cells and lymphoma tumor cells triggers the establishment of the tumor supportive niche. DLBCL, FL, and CLL display distinct patterns of BM involvement, but in each case tumor-infiltrating stromal cells, corresponding to cancer-associated fibroblasts, exhibit specific phenotypic and functional features promoting the recruitment, adhesion, and survival of tumor cells. Tumor cell-derived extracellular vesicles have been recently proposed as playing a central role in triggering initial induction of tumor-supportive niches, notably within the BM. Finally, the disruption of the BM stroma reprogramming emerges as a promising therapeutic option in B-cell lymphomas. Targeting the crosstalk between BM stromal cells and malignant B cells, either through the inhibition of stroma-derived B-cell growth factors or through the mobilization of clonal B cells outside their supportive BM niche, should in particular be further evaluated as a way to avoid relapses by abrogating resistance niches.</jats:p>	[ { "section_content": "B-cell non-Hodgkin lymphomas (B-NHL) are a heterogeneous group of hematological malignancies that emerge from different stages of normal mature B-cell differentiation (1).Lymphoma evolution and treatment are complicated by a high prevalence of relapses (2) primarily due to the ability of malignant B cells to interact with protective lymph node (LN) and bone marrow (BM) microenvironments (3)(4)(5).In agreement, several studies have correlated BM involvement with worsened prognosis and impaired chemotherapeutic response in B-cell lymphomas (6)(7)(8).This review delves into the current knowledge of the BM stromal cell modifications induced by the protumoral niche establishment in B-NHL with a specific focus on diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL).Interestingly, these three B-NHL subtypes displayed various BM involvement, with 11%-34% of DLBCL (9,10), 70%-80% of FL (11), and virtually all CLL cases showing BM infiltration at diagnosis (Table 1).Moreover, this review highlights the newly described role of extracellular vesicles (EVs) in the seeding of the BM niche.EVs are released during homeostasis and cell activation, with pleiotropic effects on signaling between cells.EV cargos are enriched in nucleic acids, proteins, and lipids.Briefly, the International Society of Extracellular Vesicles had classified EVs into three main groups: i) exosomes, the small vesicles with diameters ≤100-150 nm that are formed inside multivesicular bodies; ii) microvesicles, medium-size vesicles of plasma membrane origin with diameters of up to 1000 nm; and iii) apoptotic bodies, the large vesicles with diameters > 1000 nm that are produced by apoptotic cells (12).Excellent reviews on the biomolecular and functional characteristics of EVs as well as on the techniques used for EV isolation and characterization have recently been published (13,14). DLBCL is the most common aggressive B-NHL and accounts for approximatively 24% of new NHL cases (15).Gene expression analysis and study of genomic alterations have identified distinct genetic subtypes in DLBCL, reflecting differential pathogenesis, and associated with distinct clinical behavior (16)(17)(18)(19).Interestingly, recent studies have highlighted the impact of tumor microenvironment (TME) heterogeneity on tumor B-cell biological features and on DLBCL patient outcome (20,21). FL accounts for about 20% of adult lymphoma and is an indolent disease characterized by prolonged periods of remissions preceding relapses and ultimate transformation into DLBCL in about 30% of cases.The genetic hallmark of FL is the t (14,18) translocation occurring during the V(D)J recombination of immunoglobulin genes in the BM.The resulting deregulation of BCL2 provides a selective survival advantage to B cells during the germinal center (GC) reaction, triggering illegitimate recirculation of t (14, 18) pos post-GC B cells detectable in most healthy individuals.Iterative (re)entry of these FL precursor cells inside GC favors accumulation of additional genetic alterations sometimes converging towards overt FL (22).Importantly, FL is the paradigm of a neoplasia fully dependent on a complex microenvironment network that coevolves with tumor B cells to create a tumor supportive niche in both LN and BM (23,24). CLL is the most common hematologic malignancy in adults in Western countries.CLL is preceded by a stage of monoclonal B-cell lymphocytosis and is characterized by the accumulation of mature clonal B cells resistant to apoptosis in the blood, BM, and lymphoid organs.Patients with CLL have a heterogeneous clinical course with some never needing treatment, while others require treatment immediately after diagnosis or during illness due to a more symptomatic and unfavorable clinical course.In typical CLL cases, the tumor B cell clone exhibits an abnormal expression of markers like CD5, CXCR4, and ZAP-70, that are used to stratify the disease in conjunction with the mutational status of the BCR reflecting different cell of origin (25,26).Despite fully disseminated presentation, TME provides crucial survival signals to malignant CLL cells within the proliferation centers of LN and BM (27). In these three mature B-cell neoplasms, specialized tumor niches support survival, proliferation, and drug resistance of tumor B cells.These highly heterogeneous niches include defective tumor immunity, due to altered recruitment and cell exhaustion of cytotoxic cells, to the amplification of immunosuppressive cells, or to immune escape mechanisms developed by tumor B cell themselves, hampering tumor recognition, immune synapse formation, or anti-tumor cell activation (23,24,27).Conversely, fully functional tumor permissive cells, including CD4 pos T cell, myeloid cell, and stromal cell subsets, could be found.The relationship between LN and BM protumoral niches and how the similarities and differences between these microenvironments could impact malignant B-cell features remains elusive.In FL, malignant B cells found in the BM are characterized by a lower cytological grade, a decreased proliferation, and a reduced CD10 expression compared with LN FL B cells (28).Moreover, their gene expression profile reflects their reduced proliferation and active metabolism (29).Finally, somatic hypermutation analysis and targeted deep sequencing demonstrate that different FL B-cell subclones could be detected within LN versus BM, and suggested that FL originates in the LN and infiltrates BM early in the course of the disease, allowing further accumulation of BM-specific (28,30,31).Besides the exact cell composition and supportive signals provided by BM niches, a major issue remains to establish how these niches evolve during tumor development, from the pre-tumoral stage to overt lymphoma, during remissions and relapses. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "BM constitutes the primary site for the maintenance and differentiation of hematopoietic stem cells (HSCs) and for Bcell lymphopoiesis.Different stromal cell niches dynamically control these processes.Seminal papers have recently proposed a molecular atlas of the BM stromal cells at the single cell resolution, including osteoblasts, perivascular cells, endothelial cells, and mesenchymal stromal cells, providing clues on how various stromal cell subtypes could interact with HSCs and differentiating B-cell subsets (32)(33)(34).In the context of B-NHL, dynamic interactions between BM stromal cells and tumor B cells have been described to play a key role in converting the BM TME into a tumor supportive niche (34)(35)(36).DLBCL, FL, and CLL display distinct patterns of BM infiltration (Table 1).DLBCL show a mixed pattern of BM involvement that can potentially range from localized focal infiltrates to complete disruption of BM by lymphoma cell proliferation (37).In contrast, FL infiltration is primarily localized to the paratrabecular regions as nodular aggregates admixed with lymphoid-like TME (38).In CLL several BM infiltration patterns can be found including mixed nodular-interstitial, interstitial, and diffuse (39).In each cases, stromal cells exhibiting specific functional phenotype support recruitment, survival, and proliferation of tumor B cells, mimicking the cancer-associated fibroblasts (CAFs) described in solid cancers. ", "section_name": "LYMPHOMA BM STROMAL MICROENVIRONMENT", "section_num": null }, { "section_content": "BM DLBCL-CAFs have been poorly explored in situ.In contrast, in FL, BM-CAFs, like their LN counterparts, overexpress CXCL12 involved in the recruitment, adhesion, and activation of FL B cells (40) (Table 1).Moreover, they ectopically express CXCL13 and CCL19, the two lymphoid chemokines classically expressed by LN follicular dendritic cells (FDC) and fibroblastic reticular cells (FRC) respectively, thus recreating GC-like structures able to recruit and support CXCR5 pos CCR7 pos FL B cells (41,42). CLL B lymphocytes could be attracted in vitro to BM stromal cells whose protective effects require close cell proximity (43)(44)(45).This colocalization of CLL tumor cells with their supportive stromal cell niche relies on the deregulation of several chemokine pathways (Table 1).The demonstration that the clinical efficacy of BCR inhibitors in CLL is mediated, at least in part, by the inhibition of chemokine receptor activity and the corresponding mobilization of tumor cells out of their protective niches further highlights the crucial role of stromal cell-derived chemokine in CLL survival (46).First, high expression of CXCR4 on the surface of peripheral blood CLL cells triggers their migration to BM stromal cells producing CXCL12 (45,(47)(48)(49).CXCR4 surface expression is regulated by its ligand, thus explaining the decrease in CXCR4 expression on tissue tumor B cells, while recirculating CLL B cells express high levels of CXCR4.In parallel, blood CLL cells express high amounts of CCR7 (50).Indeed, the recycling of CXCR4 and CCR7 receptors is potentiated in CLL cells and contributes to their stronger expression (51).Recently, it was shown that p66Shc (SHCtransforming protein 1), which limits the recycling of CXCR4 and CCR7 by inhibiting their de-phosphorylation, is deficient in CLL (52).Interestingly, CCR7 could also form heterodimers with CXCR4 thus disrupting the CXCR4/CXCL12 downstream signaling and reducing B-cell retention within BM (53).Furthermore, other proteins expressed by CLL cells, such as ZAP70 or CXCR7 have been shown to regulate the function of CXCR4 (54,55).Altogether, the modulation of CXCR4 function could regulate the homing capacity of CLL cells within BM.Second, CXCR5, the CXCL13 receptor, is also expressed at high levels by CLL cells (56,57).However, conversely to the ectopic induction of CXCL13-expressing FDC in FL BM, CXCL13 seems to be only involved in CLL B cell homing into LN and the increase of CXCL13 level in the plasma of CLL patients is correlated with LN size but not BM infiltration (58).Finally, integrin a4b1 (VLA-4) plays a prominent role in the homing of CLL cells to BM niches.VLA-4 major ligands, fibronectin and VCAM-1, are constitutively present on BM stromal cells and endothelial cells and are upregulated by inflammatory signals in a NF-kB-dependent manner (59).In mouse xenograft models, CLL cells from VLA-4 neg patients showed significantly lower BM homing rates than those from VLA-4 pos patients.In contrast, the spleen homing rates did not significantly differ.Clinically, the VLA-4 status directly drives in the extent of human BM infiltration (60). ", "section_name": "BM Stromal Cells Support B-Cell Recruitment", "section_num": null }, { "section_content": "In DLBCL, the upregulation of Notch-3 in tumor cells under close cell-cell contact with BM-derived stromal cells has been implicated in the development of aggressive lymphoma cells (61).In turn, such direct interaction between DLBCL cells and stromal cells mediates an increase in B-cell activating factor (BAFF) expression by stromal thus resulting in a decrease of chemotherapy-induced B-cell apoptosis (62, 63) (Table 1).One of the factors involved in the regulation of DLBCL B-cell interaction with the BM stromal niche is the level of Jun expression.Indeed, Jun-regulated genes mediate the interaction of malignant cells with stromal cells and extracellular matrix proteins and impact extranodal localization (64).There is also evidence for tumor permissive effects of BM stromal cells on DLBCL cells through secretion of IL-6 and IL-17A, which promote both cell proliferation and drug resistance (8).Finally, the crosstalk between malignant B cells and stromal cells in DLBCL could also impact metabolic reprogramming in DLBCL.DLBCL have been early considered as metabolically heterogeneous (65,66).Non-malignant cells from TME including stromal cells have been proposed to contribute to DLBCL metabolism by providing metabolic intermediates (67) but no data specifically address this issue in BM versus LN niches even if the use of specific metabolic inhibitors have been recently explored in some DLBCL subsets (68). In FL, tumor B cells are strongly dependent on direct interactions with a microenvironment close to that of normal GC, including in particular follicular helper T cells (Tfh), myeloid cells, and lymphoid stromal cell subsets (23,24,69).The protumoral role of infiltrating lymphoid stromal cells has been demonstrated in particular by the identification of ectopicallyinduced FRC-and FDC-like cells within invaded BM (40,70).To date the origin and heterogeneity of the stromal cells supporting FL B cells within LN and BM are not perfectly understood and it is very likely that several FL CAF subtypes co-exist and organize different cell niches with specific functions (38).Stromal cells supporting FL B cell survival have been initially identified as lymphoid-like stromal cells obtained in vitro by stimulation of BM mesenchymal precursors by TNF-a (TNF) and Lymphotoxin-a1b2 (LT) or by direct contact with malignant B cells (3).Interestingly, BM stromal cells obtained from FL patients display a specific gene expression profile even after in vitro amplification, suggesting an imprinting on these cells by the tumor context (40,63,71).VLA-4, which is expressed by FL-CAFs, is involved in the growth of GC lymphomas and their resistance to anti-CD20 treatments (72).In vitro, FL stromal cells decrease tumor B cell apoptosis through a set of partially resolved mechanisms, including the production of hedgehog ligands (Hh), BAFF and TGF-b, over-expression of ABC-type multi-drug transporters, and activation of a c-MYC/HDAC6 loop in tumor cells (24,73).Moreover, CXCL12 contributes to FL B cell activation and synergize with BCR signaling (40).To date, the metabolism of FL remains broadly unexplored.Gene expression profile of FL B cells obtained from medullary niche reveals a decreased expression of the genes involved in of glycolysis, fatty acid synthesis, and OxPhos pathway compared to LN B cells (29).However, the role of stromal cells from BM versus LN niches in FL B-cell metabolic reprogramming remains to be evaluated. CLL B cells could interact with stromal cells via different receptor/ligand couples including ICAM-1/LFA-1 (74), VCAM-1/ VLA-4 (75-78), CXCR5/CXCL13 (79), BCMA/BAFF, or TACI/ BAFF (80), or by transpresentation of IL-15 from stromal cells to B cells (81).Amon those, ICAM-1, VCAM-1 and BAFF have been shown to be expressed by BM stromal cells.These interactions could lead to leukemic cell survival via a CD44-dependent mechanism involving up-regulation of MCL-1 in CLL B cells (82), activation of NF-kB pathway (80), and result in migration and proliferation of leukemic cells.In the same way, the interaction between CD100 (on CLL B-cell surface) and Plexin-B1 (present on BM stromal cells) extends CLL B cell viability and enhances proliferation (83).The mutual activation of stromal cells and tumor cells also depends on the CLL-mediated activation of Notch2 in BM stromal cells, leading to C1q overexpression the reciprocal activation of the canonical Wnt pathway in CLL cells (84) Moreover, BM stromal cell derived CXCL12 exhibits a pro-survival effect on CLL tumor cells (44,85,86).BM Stromal cells may also induce protective epigenetic modifications in CLL B cells including hypomethylation of the lysine 27 of histone H3 protein subunit (H3K27me3) (87).Finally, BM stromal cells have an important role on CLL metabolism.CLL cells have a net increase of reactive oxygen species (ROS) compared to their normal counterpart and are highly sensitive to cellular antioxydants, such as glutathione, to maintain their redox balance.BM stromal cells trigger glutathione synthesis by CLL cells through cysteine release, thus protecting tumor cells from drug-induced apoptosis (88).Moreover, BM stromal cells contribute to the glycolytic shift in CLL cells, at least in part by the Notch/Myc axis, triggering an increased glycolysis associated with higher lactic acid production, glucose uptake, and glucose transportation (89,90). ", "section_name": "BM Stromal Cells Support B-Cell Survival", "section_num": null }, { "section_content": "Beyond these functions of direct B-cell support, lymphoma CAFs are thought to be the organizers of the tumor niche.A role for the composition of the stromal-cell derived extracellular matrix in the pathogenesis of DLBCL was recently identified within tumor LN, raising the question of its direct and indirect impact on tumor growth, as an example through the modulation of immune cell infiltration, within invaded BM (21). FL-CAFs overexpress the chemokine CCL2 within invaded BM, thus triggering the recruitment of monocytes that are then converted into pro-angiogenic and anti-inflammatory macrophages (71).FL tumor-associated macrophages have been shown to play a key role in the growth of FL B cells through the transpresentation of IL-15 and the triggering of BCR-dependent signaling involving DC-SIGN-expressing macrophages and oligomannose residues introduced in FL BCR (91,92).BM and LN FL-CAFs could also promote the recruitment and survival of pro-tumoral neutrophils through the release of large amounts of IL-8 (63).Of note, in DLBCL, tumor cells have been shown to produce themselves IL-8 involved in the recruitment of APRIL-producing neutrophils (93).Moreover, BM and LN FL-infiltrating stromal cells also overexpress the immunosuppressive molecule PGE2 (94) involved in the recruitment or activation of suppressor cells such as Tregs and MDSCs (95).Finally, CAFs have been shown in solid tumors to physically hamper the recruitment of cytotoxic T cells to the tumor and CD8 pos T cells are retained at the periphery of FL tumor aggregates in both LN and BM, suggesting that FL-CAFs could contribute to tumor exclusion in lymphomas (96)(97)(98). Overall, it is clear that close interactions of tumor B cells with stromal cells within the BM, together with modulation of chemokines and cytokines directly influence the growth of DLBCL, FL and CLL, providing evidence that the BM niche plays a critical role in both lymphoma survival and drug resistance.Regardless of their cell of origin, the mechanisms underlying the differentiation of lymphoma CAFs are of the utmost importance given their potential as therapeutic targets. ", "section_name": "BM Stromal Cells Organize the Tumor Niche", "section_num": null }, { "section_content": "FL tumor B cells could directly contribute to the commitment of BM stromal precursors into an FRC-like phenotype overexpressing CCL2 and IL-8 through TNF-dependent mechanisms (3,63,71).Moreover, even if they produce less LT than normal centrocytes, the large number of GC-like B cells ectopically found in invaded FL BM probably contributes to a local overproduction of LT that synergizes with TNF for the induction of lymphoid stroma commitment.However, surrounding non-malignant cells could also participate in the polarization of FL-CAFs.Neutrophils, recruited by IL-8-producing BM FL stromal cells, could in turn contribute to their differentiation into FRC-like cells through activation of the NFkB pathway (63).In addition, LN FL-Tfh overexpress IL-4 which induces a Transglutaminase hi Podoplanin low CD106 hi CXCL12 hi phenotype on human stromal cell precursors.FL-Tfh also produce high amounts of TNF and LT, which sensitize stromal cell precursors to the effect of IL-4, notably through increased expression of the STAT6 signaling molecule (40).Even iffully mature Tfh have not been detected within FL BM, IL-4 and CXCL12 have been shown to be correlated in invaded FL BM (40).Finally, some of the recurrent genetic alterations in FL regulate the re-education of the tumor niche by tumor B cells.In particular, the gain-of-function mutations of the histone methyltransferase EZH2, which occurred early in 20% to 30% of FL, are proposed to uncouple GC B cells from the critical Tfh checkpoint whereas switching them to FDC dependency (99).EZH2-mutated GC B cells downregulate many genes linked to Tfh signaling, fail to engage Tfh, thus limiting recycling toward the dark zone of GC, and survive in the light zone as proliferating centrocytes overexpressing LT, TNF, and BAFFR, all involved in GC B-cell/FDC crosstalk.HVEM loss-of-function mutations detected in about 40% of patients with FL have been associated, in a murine model of FL and in FL patients, with an amplification of Tfh producing large amounts of IL4, TNF, and LT, and able to activate FL-CAF within LN (100).No study had currently evaluated how these genetic events could impact FL TME coevolution within BM.Even if such data are essentially lacking in the context of DLBCL, some recurrent genetic alterations have been recently associated with a specific TME pattern, with some of them related to overexpression of genes associated with GC-like stroma or extracellular matrix/FRC/CAF genes (21). Finally, LT produced by CLL cells is involved in the polarization and/or in situ generation of the tumor stromal network and the secretion of CXCL13, IL-6, and IL-8 (74,79).Moreover, the leukemic clone produces retinoic acid in the stromal microenvironment which contributes, at least in part, to the CXCL13 induction (101). In addition to the factors described above, tumor derived EVs seem to be involved in the communication between tumor cells and their TME, in particular CAFs.Such mechanism could play a central role in triggering initial induction of tumor-supportive niche within distant sites, including BM. ", "section_name": "EMERGENCE OF THE BM LYMPHOMA STROMAL MICROENVIRONMENT", "section_num": null }, { "section_content": "To date no study has explored the putative involvement of EVs in the induction of a BM lymphoma stromal niche in the context of DLBCL.Moreover, only few studies have investigated the involvement of EVs in the pathophysiology of FL (Figure 1). Recently, FL-derived EVs were shown to modulate the gene expression profile of BM stromal cells, triggering an upregulation of HSC niche factors including CXCL12, angiopoetin-1, KITLG, or IL-7, and increasing the capacity of stromal cells to interact specifically with BM FL B cells and support their survival and their quiescent phenotype (29).Interestingly, the phenotype of EVtreated stromal cells is quite different from that obtained under treatment by TNF/LT or coculture with FL B cells supporting a role of EVs in the activation of BM stromal cells before BM seeding by malignant B cells.In fact, the level of CXCL12 is increased in noninvolved BM plasma suggesting that FL EVs could shape the BM stromal niche before BM infiltration by tumor cells or at distance from this BM infiltration (unpublished data).In the same way, the analysis of the gene expression profile of BM stromal cells highlights a continuum ranging from healthy donor BM stromal cells, to stromal cells obtained from FL patients without BM involvement, and finally from FL-invaded BM (29).Altogether these data suggest that EVs could contribute to CXCL12 upregulation in the absence of direct contact with malignant B cells and could then synergize with IL-4 produced by infiltrating T cells admixed with FL B cells to further enhance local CXCL12 production.Interestingly, BM stromal cells activation by FLderived EVs was shown to rely on TGF-b dependent pathways something that is reminiscent of the role of TGF-b in the B-cell/ stromal cell crosstalk within FL LN (42).How TGF-b and STAT6 pathways could synergize for the acquisition of FL CAF phenotype within FL BM remains to be explored. Bidirectional crosstalk has also been reported between CLL Bcells and their surrounding stroma via EVs (Figure 1).CLL B cells release large amounts of exosomes that show strong expression of CD37, CD9, and CD63.Ibrutinib, a Btk inhibitor, significantly reduces the amount of plasma exosomes in CLL patients.Likewise, in vitro treatment of CLL cells with Idelalisib (a PI3K inhibitor) decreases exosome secretion, something that is not observed during treatment with fludarabine (102).This result highlights the role of the BCR-PI3K pathway in controlling exosome secretion in CLL.Besides BCR itself, CLL supportive TME produces BAFF, APRIL, CD31, and plexin B1 that all protect CLL cells from spontaneous apoptosis by synergizing with BCR signaling (44,103) and could influence EV secretion.The comparison of the mRNA content of EVs produced by B cells from healthy donors versus patients with CLL, and stimulated or not through the TLR9 pathway, shows enrichment for the kinases of the BCR pathway, LYN, SYK, MAPK1, MAPK2, and the antiapoptotic proteins BCL2 and BCL3 in CLL-derived EVs.These EVs released by tumor B cells transfer their mRNA content to non-malignant cells in the TME (104).Microvesicles derived from malignant CLL cells and detected in peripheral blood also deliver the receptor tyrosine kinase Axl into BM stromal cells leading to the activation of a AKT/mTOR/p70S6K/HIF-1a axis resulting in an increase in VEGF synthesis (105).This increase in VEGF is associated with an increased neovascularization in medullary (106) and extramedullary tissues, as well as a paracrine pro-survival stimulation of tumor B cells (107).The miRNA content of CLL B cell-derived exosomes is strongly enriched in miR-21, miR-155, miR-146a, miR-148a, and let-7g (108).BM stromal cells treated in vitro with these CLL exosomes acquire an inflammatory pro-tumoral phenotype, while endothelial cells increase their capacity for angiogenesis (108).These effects are consistent with what is known about the effect of miR-21 and miR-146a in the transition from normal fibroblast to CAFs (109)(110)(111)(112). Indeed, CLL miR-146a pos exosomes induce the transition of BM stromal precursors into CAFs showing overexpression of a-SMA and FAP (113).In addition, CLL exosomes show specific enrichment in miR-202-3p, able to decrease expression of Sufu (a component of the hedgehog pathway) in stromal cells and to trigger stromal cell proliferation (114).Finally, EVs isolated from cultures of CLL BM stromal cells induce a significant decrease in spontaneous apoptosis of tumor B cells and an increase in their chemoresistance to several drugs, including fludarabine, ibrutinib, idelalisib, and venetoclax.In addition, these EVs induce changes in the gene expression profile of CLL cells mimicking the transcriptomic signatures obtained after BCR stimulation (115). ", "section_name": "ROLES OF EVS IN THE INDUCTION OF BM LYMPHOMA STROMAL NICHE", "section_num": null }, { "section_content": "Analyzing the deregulation of extracellular proteins or miRNAs in the blood and tumor niches of patients during B cell tumorigenesis is a reliable tool for the identification of new tumor-targeted therapies.For example, the detailed mode of action of the CD30 antibody-drug conjugate Brentuximab vedotin in DLBCL is not well understood since the clinical outcome seems to be partially independent of the CD30 expression on the tumor cells.However, as CD30 pos bystander cells are enriched in the tumor tissue in many cases of DLBCL, CD30 might be released within TME-derived EVs.Thus a model was proposed in which even in the absence of CD30 on the tumor cells, EVs can transport the targeting protein from cells of the TME to tumor cells (116).This model would explain the clinical efficacy of Brentuximab vedotin also in cases of lack of the targeting antigen on tumor cells.In the same way, DLBCL EVs carrying miR-125b-5p can reduce tumor sensitivity to rituximab by inhibiting TNFAIP3 expression and reducing CD20 expression (117).Whether the miR-125b-5p/TNFAIP3 axis can be used as a therapeutic approach for increasing DLBCL sensitivity to anti-CD20 antibodies requires further investigations. EVs released by B cell could carry CD39 and CD73, two surface molecules known to hydrolyze ATP released by dying cancer cells into adenosine that hijacks CD8 T cell immune activity by binding the A2A adenosine receptors (118).One could speculate that B-cell-derived EVs may have a similar effect.The decrease of B-cell-derived EVs bearing CD73 and CD39 can be achieved by deregulating the docking protein RAB27A (118).This could be performed using an inactivated Epstein-Barr virus carrying siRNA, but it is also possible to generate EVs derived from cell lines producing RAB27A siRNA and to specifically deliver it to tumor cells. Ultimately, thanks to their molecular structure mimicking the plasma membrane of the cells and their capability to reverse their cargo into target cells, exosomes could be shaped and filled of drug molecules, acting as drug-delivery systems.In fact, cancer vaccine clinical trials relying on the administration of exosomes produced by dendritic cells (Dexosomes), exploited to shuttle antigenic determinants of immune response, were conducted to immunize patients in the context of solid tumors (119)(120)(121).In the same way, systemic administrations of TNF-Related Apoptosis-Inducing Ligand (TRAIL)-armed exosomes have shown a great anti-tumor effectiveness against FL/DLBCL cell lines both in vitro and in a mouse model (122). ", "section_name": "DISRUPTING THE EV \"REMOTE COMMUNICATION\" TO IMPROVE LYMPHOMA PROGNOSIS", "section_num": null }, { "section_content": "Despite very interesting recent data highlighting BM as a survival niche for lymphoma B cells, numerous controversies remain open on the role of the BM versus LN niches during the early step of lymphomagenesis or at the stage of post-treatment minimal residual disease that could generate relapse.In FL, both pretumoral B cells and early committed precursor cells, that will give rise to overt FL, have been shown to be enriched in BM (22).However, transformation events required iterative passages throughout the GC making it difficult to define precisely whether BM is a primary or a secondary tumor niche.The influence of tumor genetics or patient features on the capacity of tumor B cells to home and develop into BM remains completely unexplored.A major limitation for all BM-dedicated studies is the limited availability of good quality samples to perform phenotypic, transcriptomic, and functional studies and the lack of iterative sampling allowing evaluation of the impact of disease evolution or therapeutic strategies.BM aspirates are scarce and do probably not include the whole diversity of tumor/TME components, in particular stromal cells.Moreover, fixed BM biopsies are very difficult to exploit for spatial transcriptomics and even multiplex immunohistofluorescence approaches.Such technical issue hampers a precise evaluation of spatial heterogeneity in B-cell lymphomas integrating BM as a key tumor site. Altogether, many evidence support the clinical interest of targeting the crosstalk between BM stromal cells and malignant B cells, through the inhibition of stroma-derived B-cell growth factors, the mobilization of clonal B cells outside their supportive BM niche, or the reprogramming of tumor-supportive stromal cells.Identifying the best therapeutic options, and how to combine them with tumor-targeting drugs or immunotherapy approaches will be the major challenge in the field. ", "section_name": "CONCLUSION", "section_num": null } ]	[ { "section_content": "This work was supported by research grants from Fondation ARC (PGA1 RF20170205386) and the Institut National du cancer (INCA AAP PNP19-009). ", "section_name": "FUNDING", "section_num": null }, { "section_content": "ED wrote the paper, SM reviewed the paper, and KT supervised and wrote the paper.All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "ED wrote the paper, SM reviewed the paper, and KT supervised and wrote the paper.All authors contributed to the article and approved the submitted version. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. ", "section_name": "Conflict of Interest:", "section_num": null } ]
10.1038/ncomms15102	Tet2 loss leads to hypermutagenicity in haematopoietic stem/progenitor cells	<jats:title>Abstract</jats:title><jats:p>TET2 is a dioxygenase that catalyses multiple steps of 5-methylcytosine oxidation. Although <jats:italic>TET2</jats:italic> mutations frequently occur in various types of haematological malignancies, the mechanism by which they increase risk for these cancers remains poorly understood. Here we show that <jats:italic>Tet2</jats:italic><jats:sup>−/−</jats:sup> mice develop spontaneous myeloid, T- and B-cell malignancies after long latencies. Exome sequencing of <jats:italic>Tet2</jats:italic><jats:sup>−/−</jats:sup> tumours reveals accumulation of numerous mutations, including <jats:italic>Apc</jats:italic>, <jats:italic>Nf1</jats:italic>, <jats:italic>Flt3</jats:italic>, <jats:italic>Cbl</jats:italic>, <jats:italic>Notch1</jats:italic> and <jats:italic>Mll2</jats:italic>, which are recurrently deleted/mutated in human haematological malignancies. Single-cell-targeted sequencing of wild-type and premalignant <jats:italic>Tet2</jats:italic><jats:sup>−/−</jats:sup> Lin<jats:sup>−</jats:sup>c-Kit<jats:sup>+</jats:sup> cells shows higher mutation frequencies in <jats:italic>Tet2</jats:italic><jats:sup>−/−</jats:sup> cells. We further show that the increased mutational burden is particularly high at genomic sites that gained 5-hydroxymethylcytosine, where TET2 normally binds. Furthermore, <jats:italic>TET2</jats:italic>-mutated myeloid malignancy patients have significantly more mutational events than patients with wild-type <jats:italic>TET2</jats:italic>. Thus, <jats:italic>Tet2</jats:italic> loss leads to hypermutagenicity in haematopoietic stem/progenitor cells, suggesting a novel <jats:italic>TET2</jats:italic> loss-mediated mechanism of haematological malignancy pathogenesis.</jats:p>	[ { "section_content": "T en eleven translocation methylcytosine dioxygenases (TET1/2/3) catalyse the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and can further oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) [1][2][3] .5fC and 5caC can then be removed by thymine DNA glycosylase (TDG) of base excision repair (BER) 4 .Alternatively, deamination may occur at 5hmC sites by AID/ APOBEC cytidine deaminases to generate 5-hydroxymethyluracil (5hmU), which can also be repaired by BER 5 .Therefore, DNA methylation and TETs/TDG-BER-driven DNA demethylation form a complete cycle of dynamic cytosine modifications.The oxidation and demethylation of 5mC in the genome are regulated in a sophisticated manner.Genetic inactivation of Tdg and Tets leads to prominent alterations of CpG modifications at various gene regulatory regions.This raises the possibility that TETs/ TDG-BER-mediated cytosine modifications may be widespread across the whole genome. TET2 is one of the most commonly mutated/deleted genes in adult myeloid malignancies, including B30% of cases of myelodysplastic syndrome (MDS), 20% of myeloproliferative neoplasms (MPNs), 17% of de novo acute myeloid leukaemias (AMLs), 30% of secondary AMLs and 50-60% of chronic myelomonocytic leukaemias [6][7][8][9] .Somatic TET2 mutations also occur in T-cell lymphomas (such as angioimmunoblastic T lymphomas, 33%) 10 and B-cell non-Hodgkin lymphomas (diffuse large B-cell lymphoma, 12%; mantle cell lymphoma, 4%) 11,12 .Mutations in TET2 are also prevalent in healthy individuals over 70 years of age (45%) and are often associated with clonal haematopoiesis 13 .These results indicate that TET2 mutations are ancestral events that drive nonmalignant clonal outgrowth and facilitate haematological malignancy transformation.Indeed, Tet2 loss in mice leads to increased haematopoietic stem cell (HSC) self-renewal and subsequent development of myeloid malignancies [14][15][16][17] .Loss-of-function TET2 mutations and TET2 loss result in aberrant 5mC and 5hmC profiles 14,18 , and we recently showed that TET2 likely requires its catalytic activity in HSC/haematopoietic progenitor cells (HPCs) to exert a tumour-suppressive function 19 .However, the mechanisms by which TET2 loss leads to diverse haematological malignancies remain largely unknown. Accumulations of mutations in HSCs/HPCs can be deleterious to haematopoietic function and promote haematological malignancy.Here we find, using our Tet2 À / À mouse models and combined biological, bioinformatics and genetic approaches, that TET2 safeguards HSCs/HPCs against genomic mutagenicity.Exome sequencing of Tet2 À / À tumours and targeted single-cell exome sequencing of premalignant wild-type (WT) and Tet2 À / À HSCs/HPCs show that TET2 loss leads to genomic hypermutability in HSCs/HPCs.We further see that Tet2 loss leads to a significantly higher mutational frequency at genomic sites that gained 5hmC on Tet2 loss, where TET2 normally binds.Our results indicate that TET2 and TET2-mediated 5 mC oxidation safeguard cells against genomic mutagenicity.These findings suggest a novel mechanism contributing to TET2 loss-mediated pathogenesis in a diverse array of haematological malignancies. ", "section_name": "", "section_num": "" }, { "section_content": "Tet2 À / À mice develop myeloid and lymphoid malignancies.To determine the complete spectrum of haematological malignancies caused by Tet2 loss in vivo, we conducted a 2-year followup study on a cohort of 198 Tet2 À / À and 67 WT mice.All Tet2 À / À mice developed spontaneous lethal haematological malignancies with survival durations ranging from 3 to 22 months, whereas no abnormalities were detected in the haematopoietic organs of WT mice (Fig. 1a,b).Consistent with previous observations [14][15][16] , 92% of these Tet2 À / À mice developed myeloid malignancies, as indicated by monocytosis/ neutrophilia, hepatosplenomegaly and marked expansion of welldifferentiated myeloid cells or erythroid precursors in the bone marrow (BM), spleen and liver (Supplementary Fig. 1).Analyses of these 198 Tet2 À / À mice also showed that 3.5% of Tet2 À / À mice developed T-cell malignancies and 4.5% of Tet2 À / À mice developed B-cell malignancies (Fig. 1b-h, Supplementary Fig. 2 and Supplementary Tables 1 and2).These mice displayed marked lymphocytosis consisting of atypical lymphocytes, lymphadenopathy, hepatosplenomegaly and enlarged thymuses, in most cases with T-cell malignancies (Fig. 1c,d). Flow cytometric analyses of the spleen and BM cells from seven Tet2 À / À mice with T-cell malignancies revealed dominant proportions of CD3 þ T lymphocytes with a high forward scatter; five of these seven animals aberrantly expressed CD4 in their T lymphocytes that were mostly positive for CD44 and PD1 (Fig. 1e, Supplementary Fig. 2a,b and Supplementary Table 1).BM, spleen, liver and thymus had atypical lymphoid infiltrates effacing or distorting the normal architecture of these organs (Fig. 1f and Supplementary Fig. 2c).Atypical lymphocytes were medium sized, with a smaller amount of cytoplasm and irregular nuclei relative to WT.The spleen showed diffuse lymphoid infiltrates involving both red and white pulp.The liver demonstrated sinusoidal and perivascular infiltration.In addition, splenic CD3 þ cells from each of the tested Tet2 À / À mice with monomorphic T-cell infiltrations showed clonal T-cell receptor rearrangement patterns (Supplementary Fig. 2f). In nine Tet2 À / À mice with B-cell malignancies, spleen and BM cells were dominantly B220 TdT À B lymphocytes with a high forward scatter (Fig. 1g, Supplementary Fig. 2d and Supplementary Table 2).B lymphocytes from four of these mice expressed CD5 (Supplementary Table 2).Neoplastic monomorphic expansions of B cells were thus observed in these Tet2 À / À mice.Atypical lymphoid infiltrates were identified in BM, spleen, liver, lymph node and small intestine (Fig. 1h and Supplementary Fig. 2e).Atypical lymphocytes were predominantly medium sized, with a smaller amount of cytoplasm relative to WT, and with irregular nuclear vesicular and condensed nuclear chromatin.The spleen showed effacement of normal architecture with nodular lymphoid infiltrate involving predominantly white pulp.The liver displayed perivascular and sinusoidal infiltration.The intestine showed lymphoepithelial lesions with lymphoid nodules.In addition, splenic B220 þ cells from each of the tested Tet2 À / À mice with monomorphic B-cell infiltrations were clonal for IgH D-J rearrangement (Supplementary Fig. 2g). Lymphoid malignancies in Tet2 À / À mice are transplantable. To evaluate the malignant nature of the abnormally infiltrated T and B lymphocytes in Tet2 À / À mice, spleen cells from one WT mouse and two Tet2 À / À mice with malignancies, one with a T-cell clone (G3-6) and one with a B-cell clone (G3-185), were transplanted into sublethally irradiated WT recipients (Fig. 2a).No recipient receiving WT spleen cells developed any evidence of disease within 6 months of transplantation (Fig. 2b).In contrast, all mice receiving spleen cells from Tet2 À / À mice with T-or B-cell malignancy developed diseases with characteristics similar to those observed in primary mouse, for example, elevated WBC counts, lymphocytosis, splenomegaly, enlarged lymph nodes and premature death (Fig. 2b).Flow cytometric analysis of peripheral blood (PB) cells of the recipients revealed infiltration of uniform, to what we saw in the respective primary Tet2 À / À mouse (Fig. 2c,d).As Tet2 À / À B-and T-cell malignancies were transplantable into sublethally irradiated WT mice, infiltrating T or B lymphocytes in these Tet2 À / À mice are indeed malignant/neoplastic. Collectively, these data demonstrate that, in addition to myeloid malignancies, Tet2 À / À mice develop lethal T-and B-cell malignancies with features most closely resembling human peripheral T-cell lymphoma not otherwise specified and acute B-lymphocytic leukaemia, respectively.These findings are consistent with clinical observations that loss-of-function mutations in TET2 are frequent in both myeloid and subtypes of B-and T-cell malignancies [6][7][8][9][10][11]16 . Tet2 loss leads to hypermutagenicity in HSCs/HPCs.The kinetics and the involvement of multiple lineages by haematological malignancies in Tet2 À / À mice suggest that additional genetic lesions may be acquired in these mice, resulting in the pathogenesis and/or progression of various haematological malignancies.To explore this possibility, comparative genomic hybridization arrays and whole-exome sequencing (WES) were performed using tumour and non-tumour cells from Tet2 À / À mice with myeloid, T-or B-cell malignancies.Comparative genomic hybridization arrays identified a variety of structural chromosomal abnormalities in myeloid, T-and B-cell malignancies (Supplementary Fig. 3a and Supplementary Data 1).Further examination of these chromosomal deletions/gains revealed no specific regions associated with genes previously linked to tumourigenesis.WES revealed on average 10,156 nonsynonymous replacement sites and 15,809 silent sites per tumour type (Supplementary Fig. 3b and Supplementary Data 2).Of the Tet2 À / À tumours of myeloid, T-or B-cell origin, we found 190 genes with recurrent single-nucleotide variants (SNVs) originating from different tumours.The gene list included Apc, Nf1, Flt3, Cbl, Notch1 and Mll2 (Fig. 3a and Supplementary Data 3), genes recurrently altered in human haematological malignancies [20][21][22][23][24] . The heterodimerization and proline-glutamic acid-serinethreonine-rich domains of NOTCH1 are mutational hotspots in human T-ALL 24 .Notch1 mutations identified by exome sequencing and Sanger sequencing in Tet2 À / À T-cell tumours were found within these hotspots (Fig. 3b), suggesting that additional Notch1 mutations are acquired in Tet2 À / À mice and contribute to the initiation/progression of the T-cell malignancy. To confirm that mutations associated with the tumours in aged Tet2 À / À mice were somatic, we performed additional WES using Lin À c-Kit þ (LK) cells isolated from premalignant (6-week-old) WT, Tet2 þ / À and Tet2 À / À mice.WES analyses identified few mutations in premalignant LK cells from WT, Tet2 þ / À and Tet2 À / À mice, because genetic changes would only be detectable in a dominant LK cell clone (Supplementary Data 4), so variants identified in Tet2 À / À tumours are somatic mutations accumulating in Tet2 À / À cells over time, rather than germline.To overcome this limitation, we performed targeted sequencing on selected loci (from the Tet2 À / À tumour SNVs) at the single-cell level using WT and premalignant Tet2 À / À LK cells.Interestingly, we observed a significantly higher frequency of mutations on seven of the 13 selected loci in Tet2 À / À LK cells compared to WT LK cells (Fig. 3c and Supplementary Table 3), suggesting that Tet2 À / À LK cells are hypermutagenic.Higher mutational burden at sites with gained-5hmC peaks. Given the role of TET proteins in the 5mC oxidation process, we next examined the effect of Tet2 loss on genome-wide 5hmC and 5mC modification.We applied a selective chemical labelling and affinity enrichment procedure 25 to map genome-wide 5hmC distributions in premalignant WT and Tet2 À / À LK cells (Supplementary Data 5).Consistent with previous observations in mouse embryonic stem cells 26 , significant reductions of 5hmC were restricted to bodies of genes intermediately or lowly expressed in LK cells.5hmC was not affected in highly expressed genes in Tet2 À / À LK cells (Fig. 4a and Supplementary Fig. 4a).Most TET2-dependent 5hmC modifications in LK cells were located within genes (Fig. 4b). We then explored the overlap between sites with SNVs/indels in Tet2 À / À tumours and three different genomic loci: (1) sites with no change in 5hmC/5mC peaks, presumably representing the sites with 5mC oxidation catalysed by TET1/3, but not TET2; (2) sites with 5hmC peak loss or 5mC peak gain, representing the 5mC sites that are converted to 5hmC by TET2; and (3) sites with 5hmC peak gain, likely representing the 5hmC sites that are further oxidized to 5fC/5caC by TET2 (Fig. 4c).Using the w 2 test, consistent with TET2's main role being further oxidation of 5hmC, we saw a significantly greater frequency of SNVs/indels in Tet2 À / À tumours at loci with 5hmC peak gains compared to loci with no change in 5hmC/5mC peaks and loci with 5hmC peak loss or 5mC peak gain (Fig. 4d,e and Supplementary Fig. 4b,c).Similar increases were also observed with C-to-T (or G-to-A) mutations (Supplementary Fig. 4b).This mutational occurrence at loci with 5hmC peak gain was further confirmed by traditional bisulfite sequencing and TET-assisted bisulfite sequencing at CpG sites with different distances (o30 and 4100 bp) to selective mutations (detected by WES) using WT and premalignant Tet2 À / À LK cells, which showed a significantly higher percentage of CpG sites with a 5hmC gain in sites within 30 bp from the mutation sites (7 out of 10) as compared to sites 4100 bp away (1 out of 10, Po0.05, by Fisher's exact test) from the mutation sites (Supplementary Fig. 4d,e).Together, loci with 5hmC peak gain on Tet2 loss are associated with a higher mutational frequency.We next used chromatin immunoprecipitation sequencing to map genome-wide binding sites of TET2 in MEL (a mouse erythroleukaemia cell line) cells overexpressing FLAG-tagged TET2 (Supplementary Fig. 5a,b and Supplementary Data 6).Genomic analysis revealed that TET2-binding sites are enriched at regions that include exons, 5 0 -untranslated region and CpG islands (Supplementary Fig. 5c).We analysed the TET2-binding sites with differentially methylated/hydroxymethylated regions in Tet2 À / À LK cells and observed significant overlap among genes involved in myeloid and B-cell differentiation (Supplementary Fig. 5d,e).In particular, TET2 is enriched more at loci with 5hmC peak gain on Tet2 loss (Fig. 4f).Furthermore, the majority of mutations we detected within loci that require TET2 for dynamic DNA demethylation overlap with TET2-binding sites (Fig. 4g; 145 out of 212 mutation sites).Collectively, these results suggest that TET2 binds to loci marked by a gain of 5hmC on TET2 loss, and that at these sites, TET2 may protect genome stability. TET2 loss is associated with increased mutational frequency.We next examined whether TET2 loss affects the spontaneous forward mutational frequency in the hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) gene in control and TET2kd HeLa cells.The HPRT1 mutational frequency in TET2kd HeLa cells increased about 24-fold compared to control HeLa cells (Fig. 5a and Supplementary Fig. 6a,b).Similar results were obtained using control and Tet2kd NIH3T3 cells (Supplementary Fig. 6c,d).When we stably re-expressed TET2 using a lentiviral construct containing a mammalian codon-optimized cDNA sequence of TET2 into TET2kd HeLa cells (Supplementary Fig. 6e), HPRT1 mutational frequencies were completely rescued (Fig. 5a).These results indicate that TET2 loss causes increases in mutation rates.We then analysed the spectrum of mutations identified in the HPRT1 gene in 6-thioguanine (6-TG)-resistant clones of TET2kd HeLa cells.Interestingly, SNVs and singlenucleotide insertions/deletions (indels) dominated the mutations in the HPRT1 gene.Approximately 61% and 36% of the mutations in TET2kd clones were transitions and transversions, respectively, and B3% were indels (Supplementary Fig. 6f).We further analysed the mutational spectrum of Tet2 À / À tumours.We focussed on mutations located at loci with 5hmC peak gains. Base transition mutations clearly dominated the mutational spectrum in Tet2 À / À tumours, with G:C to A:T transitions accounting for 35% of total mutations.The remaining mutations consisted primarily of A:T to G:C transitions (23%), various transversions (38%), with G:C to T:A being the most frequent, and þ 1/ À 1 indels (4%), with À 1 deletions being more common than þ 1 (Fig. 5b).Of all mutations isolated, 73% occurred at CpG sites or clustered around CpG sites ( ± 30 bp); since the average distance between CpG sites across the genome is 100 bp 27,28 , o60% was expected in the gene bodies.The close association of mutation sites with CpG sites suggests a preference of mutational occurrence at or around CpG sites in Tet2 À / À tumours.The hypermutagenicity and mutational spectra of TET2kd and Tet2 À / À cells and the function of TET2/TDG in 5mC oxidation and demethylation suggest that TET2 is likely involved in safeguarding genomic mutagenicity.We further analysed a large cohort of MDS and MDS/MPN patients (Supplementary Table 4) for the presence of somatic mutations using exome sequencing of paired samples and targeted deep sequencing of 60 genes (including TET2) commonly mutated in these conditions (Supplementary Data 7).Exome analysis revealed that patients with TET2 mutations harbour a significantly higher number of mutational events compared to those with WT TET2 (Fig. 5c).Similar results were obtained in AML patient data when the The Cancer Genome Atlas (TCGA) leukaemia cohort was analysed (Fig. 5d).Of the 556 mutations identified in MDS and MDS/MPN patients with TET2 mutations, 52% and 12% were transitions and indels, respectively (Supplementary Fig. 6g), slightly higher than in patients with WT TET2 (Supplementary Fig. 6h).Confirmatory deep sequencing of a combined cohort showed that somatic TET2 mutations were associated with other subclonal events, chiefly in APC, NF1, ASXL1, CBL and ZRSR2.Consistently, many of these genes (for example, Apc, Nf1 and Cbl) are also affected by subclonal mutations in Tet2 À / À mice (Fig. 3a).Increases in numbers of subclonal events in patients with TET2 mutations are consistent with Tet2 loss leading to genome-wide hypermutability in HSCs/HPCs. ", "section_name": "Results", "section_num": null }, { "section_content": "In this study we show that, in addition to myeloid malignancies, a fraction of Tet2 À / À mice develop T-and B-cell malignancies.These findings are consistent with clinical observations that loss-of-function mutations in TET2 arise not only in human myeloid malignancies but also in subtypes of B-and T-cell malignancies [6][7][8][9][10][11]16 . Theefore, our Tet2 À / À mice can serve as a model for investigations of mechanisms by which Tet2 loss leads to diverse haematological malignancies.Leukaemogenesis is a multistep process of acquiring gene mutations.Loss-of-function TET2 mutations are initiating events in the pathogenesis of haematological malignancies.The occurrence of secondary oncogenic gene mutations, presumably in an early haematopoietic progenitor, is likely capable of modifying the disease phenotype and/or promoting the transformation/progression of a specific haematological malignancy in Tet2 À / À mice.WES identified numerous SNVs/ indels in Tet2 À / À tumours, indicating that TET2 loss constitutes a state of genetic mutagenicity.Consistently, the number of mutational events appears to be higher in MDS, MDS/MPN and primary AML patients harbouring TET2 mutations compared to those with WT TET2.However, this observation from patient exome analysis does not necessarily imply a direct role of TET2 mutations in promoting additional mutations, since other factors associated with patients with TET2 mutations might also contribute to this observation such as older ages, increased stem cell proliferation and/or longer average disease latency due to a preceding phase of clonal haematopoiesis.The increased susceptibility to mutation acquisitions is likely further accelerated in TET2-deficient HSCs/HPCs since Tet2 loss induced intrinsic changes in these cells, with increased self-renewal and proliferation. Our study identified mutations on the genes implicated in the pathogenesis of the diverse disease phenotypes in Tet2 À / À mice.Mutations in Notch1, Flt3, Nf1, Ebf1, Apc, Cbl and other genes in the Tet2 À / À haematological malignancies are consistent with human haematological malignancy gene alterations [20][21][22][23][24] .Recent studies have shown that TET2 mutations also occur in healthy elderly individuals with clonal haematopoiesis 13 .Therefore, as an ancestral event in haematological malignancies and elderly individuals, TET2 mutations represent a suitable target for intervention at the early stages of clonal expansion of HSCs/HPCs.Although we have shown that the catalytic activity of TET2 is essential for its myeloid tumour-suppressive function in HSCs/ HPCs 19 , our recent RNA-seq and 5mC/5hmC analyses on premalignant Tet2 À / À and WT LK cells showed that distinct cytosine modifications (particularly 5hmC) often mark specific genes in Tet2 À / À LK cells without altering their expression 17 .The oxidation and demethylation of 5mC in the genome are regulated in a sophisticated manner.5fC and 5caC are substrates for TDG 1,29 .TETs and TDG initiate active DNA demethylation by oxidation of 5mC and excision of 5fC/5caC in DNA, followed by incision and repair of the resulting abasic site (AP site).Although the BER process is highly accurate, errors may occur due to replication across 5mC derivatives and due to polymerase errors during gap filling.Indeed, a recent in vitro biochemical study showed that TET1 and TDG mediate sequential demethylation of symmetrically methylated CpGs to avoid DNA double-strand breaks, which could kill or transform the cells 30 .5mC is found to be a mutagen in tumour cells 31 .In addition, a recent report finds that 'G'-5caC base pairs mimic mismatches during DNA replication 32 .Thus, genomic 5mC derivatives should normally be processed error free by BER, with mismatch repair likely serving as a 'backup' for certain 5mC oxidation derivatives during DNA replication. Mutations are generally considered to occur randomly throughout the genome.Although we did not observe an overt base preference in Tet2 À / À tumours mutations, the mutations did tend to be proximal to CpG sites.Our study also demonstrated that a significantly higher frequency of mutations occurred at loci with the gain of 5hmC in Tet2 À / À tumours, where TET2 normally binds.The mechanisms that govern the association of Tet2 loss with increased local mutation rates at/around gained-5hmC sites remain to be determined.A recent study showed that Ung À / À mice exhibit higher spontaneous mutation rates and increases in spontaneous B-cell lymphomas 33 .Furthermore, Mbd4 À / À mice were recently characterized as having increased rates of C-to-T transitions at CpG sites 34 .In this backdrop, our studies suggest that TET2 loss and TET2 lossmediated 5mC oxidation dysregulation are associated with increased mutagenicity at specific genomic loci.Thus, TET2 protects HSC/HPC genomes against mutagenicity.There are now genomic profiling methods for 5fC and 5caC [35][36][37] .Given that 5fC and 5caC are directly excised by TDG/BER during active demethylation, it will be interesting to see whether sites with alterations in 5fC and 5caC marks in Tet2 À / À LK cells are preferentially associated with higher mutation rates.Parallel, genome-wide, base-resolution maps of 5mC, 5hmC, 5fC and 5caC in Tet2 À / À and WT LK cells are warranted for dissecting TET2-mediated genome-wide DNA demethylation dynamics in HSCs/HPCs and for uncovering correlations between specific 5mC, 5hmC, 5fC or 5caC alterations and the genomic hypermutagenicity caused by Tet2 loss.Indeed, consistent with our results, a recent report provides evidence that, more than TET1 or 3, a forte of TET2 is the further oxidation of 5hmC 38 . In summary, we show that TET2 loss leads to hypermutagenicity in HSCs/HPCs, preferentially at loci with the gain of 5hmC on the loss of TET2, where TET2 normally binds.TET2-dependent 5hmC marks are highly enriched at gene bodies, such as exons of LK cells.TET2-deficient HSCs/HPCs that become hypermutagenic are likely not malignant per se, but higher mutation rates in these cells may result in additional driver mutation(s) in TET2 target genes over time.Such states may be amenable to TET2 activity-boosting chemoprevention approaches.Our results unveil a novel role for TET2 in safeguarding genome mutagenicity and provide additional insights into the mechanisms by which loss-of-function TET2 mutations cause diverse human haematological malignancies.Further mechanistic studies are needed to determine how TET2 loss leads to increased DNA mutagenicity in HSCs/HPCs and thus the increased risks of haematopoietic malignancies. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Analyses of mice.Tet2-knockout (Tet2 À / À ) mice were generated as described 14 .Animal care was conducted in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee (IACUC), University of Miami Miller School of Medicine.PB was collected by retro-orbital bleeding of mice and was smeared for May-Gru ¨nwald-Giemsa staining, and/or subjected to an automated blood count (Hemavet System 950FS).For histopathology analyses, femurs were fixed in formaldehyde, decalcified and paraffin embedded.Spleens, livers, lymph nodes, thymus and intestine were treated similarly, except for the decalcification step.Sections (4.5 mm) were stained with haematoxylin and eosin (H&E).For flow cytometric analyses, single-cell suspensions from BM, spleen, liver, lymph node, thymus and PB were stained with panels of fluorochrome-conjugated antibodies.Dead cells were excluded by 4,6-diamidino-2-phenylindole staining.Analyses were performed using a BD FACSCanto II or LSRII flow cytometer.All data were analysed by FlowJo7.6 software. Mouse exome sequencing.Initial WES was carried out to identify candidate mutations in the exome of genes.Genomic DNA was captured with the NimbleGen mouse exome array according to the manufacturer's protocol, and 100-bp paired-end sequencing was performed using an Illumina HiSeq 2000.Raw sequencing reads were mapped to the whole mouse genome (mm10) using PEMapper/PECaller (https://github.com/wingolab-org/pecaller)with the default settings 39 , and variant bases were annotated with SeqAnt (http://seqant.genetics.emory.edu/) 40.For samples subjected to mutation detection, genomic DNA was amplified in selected exons by PCR (primers shown in Supplementary Data 8) and sequenced by Sanger sequencing.The location and types of mutations were then determined by sequencing results. HPRT mutation analyses.Mutation analyses can be achieved in proliferating cells in vitro by anHPRT1 assay that positively selects for HPRT-deficient mutants based on their resistance to 6-TG, which is lethal to HPRT-WT cells that are proficient in free purine base salvage 41 .The HPRT mutation assay was conducted as described previously 42 .Briefly, cells (5 Â 10 5 ) were seeded in triplicate in 10-cm petri dishes for 12 h and fed with complete medium containing 5 mM freshly prepared 6-TG.Plating efficiency was determined by culturing 5 Â 10 2 cells in the absence of 6-TG.After 10 days of culturing, colonies were visualized by staining with 0.05% crystal violet.The mutation frequency was then the ratio of the number of clones in the presence of 6-TG to the total number of cells plated, normalized by the plating efficiency.Types of mutations were characterized by DNA sequencing coding regions of the HPRT gene using primers shown in Supplementary Data 8. Patients.The mutational statuses for TET2 and other coexisting genes were analysed in BM and blood specimens from patients with various myeloid neoplasms, including MDS, MDS/MPN and secondary AML (see Supplementary Table 4 and Supplementary Data 5).Informed consent was obtained according to protocols approved by the institutional review boards and in accordance with the Declaration of Helsinki.Diagnosis was confirmed at each institution according to the World Health Organization classification criteria.Analysis of TCGA primary AML cases was performed using publically available data sets (http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp). WES for human patient samples.WES and targeted capture sequencing were performed as described previously 43 .For WES, the 50 Mb of protein coding sequences was enriched from total genomic DNA by liquid-phase hybridization using SureSelect (version 4) (Agilent Technology), followed by massively parallel sequencing with HiSequation 2000 (Illumina).Somatic mutations were detected using our in-house pipeline, followed by validation using amplicon deep sequencing 43,44 .To minimize false positives and focus on the most prevalent or relevant somatic events, we implemented a rational bioanalytic filtering approach and applied heuristic bioanalytic pipelines.We used two independent pipelines to identify somatic and germline alterations.For confirmation of somatic mutations, we analysed paired germline DNA from CD3 þ lymphocytes.The selected observations were validated by targeted deep sequencing using MiSeq.Our sequence library for deep sequencing was generated by TruSeqCustom Amplicon (Illumina). Targeted multiamplicon deep sequencing of patient samples.We applied multiamplicon-targeted deep sequencing (TrueSeq; Illumina) to frequently affected exons of 60 selected genes 45 .The sequencing libraries were generated according to an Illumina paired-end library protocol and subjected to deep sequencing on MiSeq (Illumina) instrumentation according to standard protocol.Highprobability oncogenic mutations were called by eliminating sequencing/mapping errors and known/possible single-nucleotide polymorphisms based on available databases and frequencies of variant reads.Genomic copy number status was calculated by directly enumerating corresponding sequencing reads in each exon. Statistical analysis.Differences between experimental groups were determined by the Student's t-test, Fisher's exact test, Wilcoxon's rank-sum test and/or analysis of variance, followed by Newman-Keuls multiple comparison tests as appropriate.P values o0.05 were considered significant.For SNV count data, w 2 tests were used as implemented in R (http://cran.r-project.org/) 46. ", "section_name": "Methods", "section_num": null } ]	[ { "section_content": "This work was supported by grants from the NIH (HL112294 to M.X., CA172408 to M.X. and F.-C.Y., NS05163, NS079625 and HD073162 to P.J. and DK110108 to S.M.), the Department of Veterans Affairs (BX001820 to T.S.W.), and national natural science foundation of China (81629001 and 81670102 to Z.Z.) ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Data availability.Genome-wide data sets generated for this study are deposited at GEO under the accession number GSE74390.All other remaining data are available within the article and Supplementary Files, or available from the authors on request. ", "section_name": "", "section_num": "" }, { "section_content": "Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.This work is licensed under a Creative Commons Attribution 4.0 International License.The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/r The Author(s) 2017 ", "section_name": "Author contributions", "section_num": null } ]
10.3390/cancers13020284	Hematopoietic versus Solid Cancers and T Cell Dysfunction: Looking for Similarities and Distinctions	<jats:p>Cancer cells escape, suppress and exploit the host immune system to sustain themselves, and the tumor microenvironment (TME) actively dampens T cell function by various mechanisms. Over the last years, new immunotherapeutic approaches, such as adoptive chimeric antigen receptor (CAR) T cell therapy and immune checkpoint inhibitors, have been successfully applied for refractory malignancies that could only be treated in a palliative manner previously. Engaging the anti-tumor activity of the immune system, including CAR T cell therapy to target the CD19 B cell antigen, proved to be effective in acute lymphocytic leukemia. In low-grade hematopoietic B cell malignancies, such as chronic lymphocytic leukemia, clinical outcomes have been tempered by cancer-induced T cell dysfunction characterized in part by a state of metabolic lethargy. In multiple myeloma, novel antigens such as BCMA and CD38 are being explored for CAR T cells. In solid cancers, T cell-based immunotherapies have been applied successfully to melanoma and lung cancers, whereas application in e.g., breast cancer lags behind and is modestly effective as yet. The main hurdles for CAR T cell immunotherapy in solid tumors are the lack of suitable antigens, anatomical inaccessibility, and T cell anergy due to immunosuppressive TME. Given the wide range of success and failure of immunotherapies in various cancer types, it is crucial to comprehend the underlying similarities and distinctions in T cell dysfunction. Hence, this review aims at comparing selected, distinct B cell-derived versus solid cancer types and at describing means by which malignant cells and TME might dampen T cell anti-tumor activity, with special focus on immunometabolism. Drawing a meaningful parallel between the efficacy of immunotherapy and the extent of T cell dysfunction will shed light on areas where we can improve immune function to battle cancer.</jats:p>	[ { "section_content": "Roughly a decade ago the interplay between malignant and immune cells has been recognized as an emergent hallmark of cancer [1].We are already witnessing the advent in the clinic of multiple strategies aimed at addressing this cross-talk and reinforcing immunity in various cancer types.The success of those therapies, generally referred to as 'immunotherapy', is dampened by the way cancer cells suppress the immune system, particularly T cells, for their own sustenance.Despite the fact that T cell dysfunction is reported to occur in many hematopoietic and solid cancers, broad mechanistic understanding and overview across the various cancer types is lacking. The full repertoire of innate and adaptive immune cells plays crucial roles in the pathogenesis of neoplastic diseases, with T cells being considered special players in cancer progression [2] due to their prominent role in destroying pre-malignant cells that could develop into cancer.The onset of an anticancer immune response is a stepwise process known as cancer-immunity cycle [3] (Figure 1), characterized by a series of events that must take place and iteratively self-propagate.Reaching a proper immune response to cancer and tolerance to self-antigens is a crucial balance that has to be maintained in order to prevent autoimmunity.Tolerance is a physiological mechanism in immunity that acts in two separate niches [4], the thymus and the so-called periphery, at different moments during the onset of an immune response.After migrating from the bone marrow to the thymus, the T cells repertoire undergoes the process of central deletion, for the elimination of self-reactive T cells by negative selection.This process of central tolerance is not completely efficient, so additional tolerance is required in the periphery.Peripheral tolerance can be achieved intrinsically via mechanisms regulating the state of T cells (anergy, apoptosis or phenotype skewing) or extrinsically, controlled by the dialogue with other cell types in this second niche, like regulatory T cells (Tregs), dendritic cells (DCs) or myeloid cells [4]. Cancer cells and the tumor niche aid T cell tolerance: immune cells can be subverted to a tolerogenic, inert and dysfunctional state by cancer cells at every level of the cycle, by means of physical interactions and soluble mediators in the immunosuppressive tumor microenvironment (TME), (Figure 1). Cancers 2021, 13, x FOR PEER REVIEW 2 of 20 in the clinic of multiple strategies aimed at addressing this cross-talk and reinforcing immunity in various cancer types.The success of those therapies, generally referred to as 'immunotherapy', is dampened by the way cancer cells suppress the immune system, particularly T cells, for their own sustenance.Despite the fact that T cell dysfunction is reported to occur in many hematopoietic and solid cancers, broad mechanistic understanding and overview across the various cancer types is lacking. The full repertoire of innate and adaptive immune cells plays crucial roles in the pathogenesis of neoplastic diseases, with T cells being considered special players in cancer progression [2] due to their prominent role in destroying pre-malignant cells that could develop into cancer.The onset of an anticancer immune response is a stepwise process known as cancer-immunity cycle [3] (Figure 1), characterized by a series of events that must take place and iteratively self-propagate.Reaching a proper immune response to cancer and tolerance to self-antigens is a crucial balance that has to be maintained in order to prevent autoimmunity.Tolerance is a physiological mechanism in immunity that acts in two separate niches [4], the thymus and the so-called periphery, at different moments during the onset of an immune response.After migrating from the bone marrow to the thymus, the T cells repertoire undergoes the process of central deletion, for the elimination of self-reactive T cells by negative selection.This process of central tolerance is not completely efficient, so additional tolerance is required in the periphery.Peripheral tolerance can be achieved intrinsically via mechanisms regulating the state of T cells (anergy, apoptosis or phenotype skewing) or extrinsically, controlled by the dialogue with other cell types in this second niche, like regulatory T cells (Tregs), dendritic cells (DCs) or myeloid cells [4]. Cancer cells and the tumor niche aid T cell tolerance: immune cells can be subverted to a tolerogenic, inert and dysfunctional state by cancer cells at every level of the cycle, by means of physical interactions and soluble mediators in the immunosuppressive tumor microenvironment (TME), (Figure 1).Before continuing, in order to better understand how cancer cells impact on each step of the process, it is beneficial to briefly recap what must happen to elicit a successful immune response to cancer.First of all, the genetic aberration(s) leading to oncogenesis generate neoantigens for tumor-specific T cell responses [3].In order to prime antigen-specific T cells to exert an anticancer response, antigen presentation (signal 1) must be accompanied by other signals: co-stimulation (signal 2) and cytokines (signal 3), (Figure 2).Only if the three signals are delivered, T cells can become activated, start to proliferate and differentiate into effector and memory cells.Following activation, co-stimulatory and co-inhibitory receptors are expressed on the cell surface.The physiological role of inhibitory receptors, also referred to as checkpoints, is to prevent autoimmunity and control the immune response.We propose that these could represent \"signal 4\" during T cell activation, given their role in maintaining T cell response homeostasis.Finally, T cells traffic to the tumor bed and infiltrate the tumor tissue, where activated T cells kill cancer cells [5] via Fas-Fas ligand interaction or by secreting cytotoxic granules containing granzyme and perforin.Various mechanisms can induce tolerance in the TME and limit the effectiveness of the immune response.As will be discussed later, peripheral tolerance is mostly observed in solid tumors, while hematopoietic malignancies show more unique defects in the regulation of central tolerance and priming [6], Table 1. Cancers 2021, 13, x FOR PEER REVIEW 3 of 20 Before continuing, in order to better understand how cancer cells impact on each step of the process, it is beneficial to briefly recap what must happen to elicit a successful immune response to cancer.First of all, the genetic aberration(s) leading to oncogenesis generate neoantigens for tumor-specific T cell responses [3].In order to prime antigen-specific T cells to exert an anticancer response, antigen presentation (signal 1) must be accompanied by other signals: co-stimulation (signal 2) and cytokines (signal 3), (Figure 2).Only if the three signals are delivered, T cells can become activated, start to proliferate and differentiate into effector and memory cells.Following activation, co-stimulatory and co-inhibitory receptors are expressed on the cell surface.The physiological role of inhibitory receptors, also referred to as checkpoints, is to prevent autoimmunity and control the immune response.We propose that these could represent \"signal 4\" during T cell activation, given their role in maintaining T cell response homeostasis.Finally, T cells traffic to the tumor bed and infiltrate the tumor tissue, where activated T cells kill cancer cells [5] via Fas-Fas ligand interaction or by secreting cytotoxic granules containing granzyme and perforin.Various mechanisms can induce tolerance in the TME and limit the effectiveness of the immune response.As will be discussed later, peripheral tolerance is mostly observed in solid tumors, while hematopoietic malignancies show more unique defects in the regulation of central tolerance and priming [6], Table 1.All cancers types induce immune dysfunction, as indicated in the first column, which lists similar aspects of immune defects.The distinctive aspects in solid versus B cell malignancies are summarized in their respective columns and addressed in further detail in the text. T cell development and differentiation is shaped and affected by the tumor [7].Naïve CD4+ (helper) or CD8+ (cytotoxic) lymphocytes differentiate into short lived effector cells upon encountering the antigen.A subset of memory cells remains long term in secondary T cell development and differentiation is shaped and affected by the tumor [7].Naïve CD4+ (helper) or CD8+ (cytotoxic) lymphocytes differentiate into short lived effector cells upon encountering the antigen.A subset of memory cells remains long term in secondary lymphoid organs or in the tumor bed.It has been widely reported that the individual patient's T cell profile has an impact on adoptive T cell therapy efficacy [8,9]: chronic antigen exposure and stimulation imposed by cancer leads to the accumulation of terminally differentiated effector T cells, while the memory phenotype is more desirable due to its enhanced persistence and increased activity [10]. As a reflection of the events regulating anticancer immunity, immunotherapy can use at least three sites for intervention: promoting priming, shifting the balance to anti-tumor T cell responses, and counteracting immunosuppression in the TME (Table 2).Immunotherapeutic strategies are broadly classified on the basis of the stage in the cycle where the intervention is attempted: vaccination, adoptive transfer of immune effectors, such as bispecific antibodies or tumor specific T cells with memory phenotype, and immunomodulatory therapy with either checkpoint blockade or cytokines [11,12].Cytokines and cytokine blockade Vaccination strategies will not be discussed here as we focus on T cells, except to state that we are currently witnessing a revival of cancer vaccines, especially due to increasing knowledge of neoantigens [13].While in the past the approach did not seem to be impactful on cancer treatment, preclinical data are nowadays showing promising results in combining vaccines and immune checkpoint therapy [14], especially when no pre-existing tumour immunity is present.Adoptive T cell therapy requires the isolation of tumor reactive lymphocytes from the patients, their expansion ex vivo in the presence of growth factors, and their re-infusion into patients.Another approach to generate tumor-specific T cells utilizes patient-derived lymphocytes engineered to express a chimeric antigen receptor (CAR) through which they are redirected to recognize the tumor.CARs are chimeric constructs consisting of an antibody region fused to a T cell receptor (TCR) signaling domain, with additional co-stimulatory domains providing signal 2. This results in T cells that are activated and proliferate in vivo upon contact with their antigen but bypassing the need for antigen processing and MHC restriction, since CAR T cells recognize the intact surface antigen.Lastly, therapeutic blockade of checkpoint pathways, administering immune checkpoint inhibitors (ICIs), aims at \"removing the breaks\" [15] imposed by the expression of inhibitory receptors on T cells. In general, the mechanisms of resistance to T cell based immunotherapy are overlapping with those used at first by cancers to evade immune destruction and initiate tumor progression [16].As a consequence, a deep understanding of cancer-induced T cell dysfunction among different cancer types might be the key to more effective therapeutic strategies.Identification of mechanisms associated with cancer-induced T cell dysfunction is crucial to understand predictive prognostic factors for therapy, improve current ICIs regimens using combinatorial approaches and generating CAR T cells that can persist and effectively eliminate tumor cells. In order to explore T cell dysfunction in cancer, we will address first the efficacy of T cell-based therapies, such as checkpoint blockade and transfer of cellular immune effectors into patients.We aim at mapping areas of similarities and differences in solid and hematopoietic tumors, with the premise that immunotherapy efficacy depends on the state of T cells in cancer patients.Here, T lymphocytes are characterized by features of cancer-induced anergy, senescence and/or exhaustion.Anergy is caused by insufficient co-stimulatory signals during priming; senescence is growth arrest after extensive proliferation; exhaustion is characterized by expression of inhibitory signals after an excessive activating signal due to chronic antigen stimulation [17].Reversing those features is the key to reach immune reinvigoration towards long-lived T cells that can quickly proliferate and renew themselves upon antigen re-encounter, such as stem cell-like memory T cell precursors. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "The success of T cell based immunotherapy in solid tumors is limited to checkpoint blockade therapies, with adoptive cell therapy lagging behind [18,19].Anticancer strategies targeting CTLA-4, PD-1, PD-L1 demonstrated clinical efficacy in many cancer types [20] leading to several FDA-approved antibodies for treating melanoma [21,22], lung carcinoma (mainly, non-squamous non-small-cell lung carcinoma, NSCLC) [23] and DNA-mismatch repair-deficient cancers [24,25].Despite its success, therapy using ICIs also encounters resistance with mechanisms that resemble those adopted by cancer cells during the initial escape phase of immunoediting [16], ultimately leading to tumor progression. Loss of immunogenicity is the first mechanism of escape that cancer cells adopt against an immune response.In general, a higher number of identified neoantigens together with more CD8+ tumor infiltrating lymphocytes (TILs) correlate with increased patient survival [26].Since neoantigens directly reflect the genomic instability of the cancer [16], tumor mutational burden (TMB) has been measured across different cancer types.Tumors with higher TMB (melanoma, lung cancer) are more likely expressing neoantigens recognizable by T cells and more likely responsive to ICIs.Nevertheless, tumor reactive T cells can be also present in cancers with relatively low TMB, such as breast cancer, which suggest that progressing malignant clones are able to escape immunity despite the presence of TILs. Loss of immunogenicity has obvious negative consequences for adoptive CAR T cell therapy as well: the search for tumor specific antigens is still ongoing in solid tumors.Unlike B cell malignancies in which the tumor cells express the B-cell marker CD19, solid tumors rarely express one tumor specific antigen [27].They are rather enriched for tumor associated antigens like mesothelin, MUC1 in melanoma, HER2 in breast cancer or express neoantigens, aberrant protein translated by genes with somatic mutations, or proteins which are abnormally expressed in cancer cells (MAGE family, melanoma associated protein).The obvious adverse effect of an antigen co-expressed in non-malignant cells is the off-target effect toxicity and efforts are being made to improve safety, such as co-expressing suicide genes in the CAR construct [28].Moreover, only a subset of tumor cells might express the antigen, and when it is uniformly expressed, antigen escape might occur: cancers hijack immune recognition through selective outgrowth of cells able to lose immunogenic antigens.Tandem CARs have been generated to address antigen heterogeneity and antigen loss, such as a dual HER2-MUC1 construct that showed promising results in an in vitro model of breast cancer [29]. Another hurdle faced by immunotherapy in solid cancers is tumor infiltration.Physical barriers can prevent TILs from infiltrating the TME [30] and this has a negative impact on the efficacy of ICIs.Stroma and abnormal immature vessels at the core of the tumor have an impact on TILs infiltration, and they act as a barrier for an infused CAR T cellular product [27].For this reason, an opportunity to enhance CAR T cell efficacy is represented by \"armored\" CAR T constructs, expressing homing receptors or chemokine, such as the anti-mesothelin CAR T construct constitutively expressing the cytokine IL-7 and the chemokine CCL19 (Figure 1) to guide the infused product to the tumor site.This construct showed complete tumor regression in a solid tumor mouse model [31]. When the cancer is immunogenic and T lymphocytes manage to infiltrate the tumor, ICIs seem still the most promising therapy and it is achieving remission in patients that a few years ago were deemed as incurable.Indeed, studies reported that in solid tumors ICIs are more efficient in patients showing TIL infiltrates [32].An intriguing finding that requires further study is that the number of circulating CD8+ T cells is negatively associated with the durable effect of ICIs in NSCLC [33], possibly reflecting tumor T cell infiltration.However, our knowledge of the role of pre-existing immunity in the efficacy of checkpoint blockade is still very limited. Adoptive immunotherapy using TILs or CAR T cells seems to require further improvements, for several reasons.Tumor infiltrating lymphocytes are dysfunctional and exhausted, due to chronic stimulation and their coping mechanisms to survive the microenvironment at the tumor site.Thus, the obvious limitation to TIL adoptive cell therapy is the expansion of sufficient numbers of reactive T cells in vitro, because TILs might be already terminally differentiated due to chronic stimulation in the TME.T cell phenotypes within the tumor do not mirror the immune composition in peripheral blood [34], where T cells do not show such an exhausted phenotype, as exemplified by a study conducted in melanoma-patients where the ratio and absolute numbers of CD4+ and CD8+ T cells in peripheral blood were found comparable to age-matched healthy donors [35], as well as the frequencies of memory T cell subsets, which did not show increased expression of inhibitory receptors.Those findings seem promising for the generation of CAR T cells for patients with solid tumors since peripheral lymphocytes seem better equipped to attack cancer cells.However, it is important to take into account that these peripheral T cells might not express the right homing receptors to reach the tumor, so they should be \"armored\" as previously indicated.A second challenge is represented by the higher proportion of Tregs found in the peripheral blood of solid malignancies, including melanoma, lung and breast cancer [35,36] that may negatively influence CAR T cell composition [37]. Even when the generation of a CAR T cell infusion product is successful and able to reach the tumor site, those cells have to face the TME.The TME is inhabited by stromal cells and suppressive immune cells including myeloid-derived suppressor cells (MDSCs), tumor associated macrophages with M2 phenotype and Tregs.Together with malignant cells, those cell types express ligands for inhibitory receptors on T cells and secrete soluble factors impacting T cell priming, survival and function, which we will discuss later.Microenvironmental clues also matter in hematopoietic tumors. ", "section_name": "Immunotherapy for Solid Tumors", "section_num": "2." }, { "section_content": "Hematopoietic malignancies develop in the same niche in which immune responses are generated, suggesting that these tumors fail to trigger immune sensing mechanisms, or effectively impair anti-tumor immune responses when they do take place [6].T cell priming usually spontaneously occurs at least in a subset of solid cancers and it is then followed by functional impairment at the tumor site.T cell tolerance in hematological malignancies is regulated at the central level, where tolerogenic antigen-presenting cells lead to a defective priming upon initial antigen encounter.Ineffective priming could be one of the reasons why ICI are less efficient in hematopoietic malignancies when compared to the successes in solid tumors, since the lack of a proper immune response does not derive from checkpoints preventing the killing of cancer cells, but rather by a state of immunological ignorance.Moreover, later on we will discuss whether T cells in multiple myeloma (MM), for example, exhibit the features of senescence or anergy, rather than exhaustion [38][39][40].This distinction could provide a second reason for the failure of ICIs in hematopoietic malignancies.ICIs showed some success in Hodgkin lymphoma [41] using the anti-PD-1 nivolumab, but have to make an impact yet in other hematopoietic cancers such as chronic lymphocytic leukemia (CLL), MM or diffuse large B-cell lymphoma (DLBCL) [42].Interestingly, the classical Hodgkin lymphoma is characterized by genetic alterations resulting in constitutive expression of PD-1 ligands [43], explaining the reason of this success.In other hematopoietic malignancies, trials are still ongoing, testing combinatorial approaches [5] with either conventional cytotoxic agents or monoclonal antibodies (mAbs) such as the anti-CD20 rituximab [44]. To date, the major successes of CAR T cell therapies have been recorded in hematological tumors [45][46][47].The first FDA approvals were witnessed in 2017, with Kymriah ® (Novartis, Basel, Switzerland) and Yescarta ® (Kite Pharma, Santa Monica, CA, USA) targeting the B cell antigen CD19 to treat B-acute lymphocytic leukemia (B-ALL) and DLBCL, respectively.CAR T cell products showed high efficacy in high-grade malignancies, but not in low-grade hematopoietic cancers.The pathogenesis of those two categories of tumors is different: high-grade tumors, such as B-ALL, are fast growing and characterized by immature precursors, while low-grade tumors, like CLL, show a slower growth of mature B cells.This subtle and persistent growth induces chronic stimulation, a state of cancer-induced T cell dysfunction and acquisition of a terminally differentiated phenotype.T cell defects in CLL due to disease and/or therapy impair ex vivo expansion and response to CAR T cells.Previous therapies for CLL, including alkylators and fludarabine, have a profound negative effect on T cell function, exacerbating the T cell defect in CLL.The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib, instead, may potentially improve T cell immunity [48] and may be administered in combination with CAR T cell therapy or ICIs [49].A recent trial using lymphodepletion and CD19 CAR T cells showed very promising results in CLL-patients after failure of Ibrutinib treatment [50], most likely because of the effect of this drug on the T cell compartment.Concerning the combinatorial approach of Ibrutinib with ICIs such as Nivolumab, an early phase trial [49] showed that the combination had no additional efficacy compared to single agents therapies in patients with relapsed/refractory CLL and other hematopoietic malignancies. In order to compare the hurdles of adoptive T cell therapy in hematopoietic cancers with the ones described for solid tumors, we will first discuss antigen selection and immunosuppressive nature of TME.While finding an optimal CAR T cell target is still a challenge in solid cancers, CD19 appears as a highly suitable target in B cell hematologic malignancies for several reasons: its expression is ubiquitous on malignant and normal B cells.Loss of normal B cells can be overcome by immunoglobulin replacement therapy [37].A more serious concern is neurotoxicity associated with CD19 targeting, most likely deriving from CD19 expression on mural cells pivotal in maintaining the integrity of brain-blood barrier [51].This off-target effect makes CD19 a less ideal target than previously expected.Moreover, as previously discussed, a second downside is that antigen escape, widely accountable for CAR-T cell resistance, has been reported with CD19 CAR as well, in B-ALL [52] and other B cell malignancies [53,54].Therefore, other targets are currently being explored, such as B-cell maturation antigen (BCMA) in MM [55] or CD22 in B-ALL [56].The approach of dual or tandem CAR has been pursued for hematopoietic cancers as well, and some constructs targeting CD19/20 or CD19/CD22 entered clinical trials in hematological malignancies [57]. Regarding the immunosuppressive feature of the tumor niche, hematopoietic cancers can be compared to solid tumors when considering that the solid component is still significant in leukemia, lymphoma or myeloma, where secondary or primary lymphoid organs function as the TME.MM is homing in the bone marrow and like in solid tumors, T cells at the tumor site are more severely impaired than the ones in peripheral blood [38].Those compartments are inhabited by immune cells, whose function, rather than eliminating leukemic cells, is redirected to promote cancer cells survival and to generate an immunosuppressive and protumorigenic microenvironment [58,59].Profound changes in the immune compartment are reported to occur in all hematopoietic cancer types, due to the fact that malignant cells themselves may be part of the immune reaction in different ways and can alter the rest of the players in adaptive immunity.For this reason, it might be speculated that tumor-host interactions in B cell tumors play a special role in tumor initiation and progression.Moreover, cancers like MM or lymphoma are homing in niches in which T cell development take place.Together, those findings suggest indeed that the immune compartment is dysfunctional since the onset of the neoplastic disease in hematopoietic malignancies, raising the \"chicken-egg\" dilemma: which comes first, cancer initiation or immune dysfunction? Compared to solid cancers, hematopoietic malignancies show more profound abnormalities in T cell development and differentiation, which are affecting also the blood compartment.Multiple myeloma patients show quantitative and functional T cell abnormalities [60], with a decrease in the CD4+/CD8+ ratio [61] associated with disease progression [62].T cell subsets skewed in favor of an immunosuppressive state [63], as shown by an increase ratio of Tregs when compared to the inflammatory T helper 17 (Th17) subset.Data suggests that the acquired T cell dysfunction derives from the specific interaction of myeloma cells with T cells resulting in impaired T cell immunity against the tumor, but the T cell response against external antigens is mostly not affected [64].Clonally expanded T cells are present in the blood of myeloma patients, with an incidence between 50-75% [40].Those clones are functionally resembling more a senescence phenotype rather than exhaustion [40], which may also explain why checkpoint blockade using PD-1 or CTLA-4 inhibitors was not successful in trials.In a study of long-term survivors of MM, those clonally expanded T cells show regained proliferative capacity, suggesting that this senescent-like state is reversible.Moreover, survivors also showed a normal or reduced Treg/Th17 ratio [65]. Immune dysregulation is also prominent feature of CLL from its early stage and alterations within the T cell compartment become more apparent with disease progression or after treatment [66].The absolute CD4+ and CD8+ counts are increased, with CD8+ subset showing a higher relative increase which accounts for the reduction of the CD4+/CD8+ ratio [67][68][69].It has been speculated that the expansion of T cell counts would represent an attempted specific immune response against CLL [66], yet CLL-specific T cells have not been reported so far.Together with quantitative changes, CD8+ and CD4+ T cells also show functional defects [70], with a marked alteration in helper activity [71].Another factor likely contributing to immune suppression is the presence of an increased proportion of Tregs [72]. There are few studies regarding the impact of T cell dysfunction in B-ALL development and relapse after (CAR T cell) therapy, and these focused mostly on the bone marrow microenvironment [73,74], which is where this malignancy of immature progenitor B cell blasts initially takes place.It has been shown that B-ALL is associated with loss of CD8+ T cells, as indicated by a significantly lower frequencies of CD8+ T cells in the patients' bone marrow compared to healthy controls, together with accumulation of late stage effector CD4+ T cells [73].These data indicated that the composition of bone marrow T cells is profoundly affected by leukemic blasts, provoking a late-stage differentiation.Studies on peripheral blood of B-ALL patients showed again an accumulation of the Treg subset as compared to healthy donors [75].As previously stated, an increased frequency of Tregs in peripheral blood is also a common feature of solid tumors, indicating that the presence of this immunosuppressive subset might play a common pivotal role in cancer progression.In the next sections we will gradually abandon the separation between solid and hematopoietic cancers and discuss more deeply contact-dependent interactions, as well as soluble factors by which tumors cells and/or other bystander cells are affecting T cell development and functions in the context of cancer-immunity-cycle (Figure 1). ", "section_name": "Immunotherapy in Hematopoietic Malignancies", "section_num": "3." }, { "section_content": "As hypothesized by Curran et al. [6], tumor specific T cells are primed but become functionally impaired at the tumor site in solid tumors.By contrast in leukemia or lymphoma T cells are not properly activated, but rather energized upon antigen encounter.This notion is supported by data collected from a murine model of acute leukemia [76], in which the implantation of leukemic cells in situ to mimic solid tumor induced an antigen-specific CD8+ T cell response while the systemic engraftment of leukemic cells induced a T cell tolerogenic state, characterized by defect in proliferation.The tolerant phenotype could be abrogated when administrating an agonistic anti-CD40 antibody, suggesting a defect on antigen presentation and antigen presenting cell (APC) activation as drivers of the tolerogenic state [76].This approach was actually implemented in B-ALL-patients after allogeneic stem cell transplantation, where it was able to induce immune responses reactive against leukemic blasts [77].Other indications of the feasibility of targeting CD40-CD40L interactions come from a phase I study conducted in CLL patients [78], where preliminary clinical responses such as the reduction of leukemic cell counts were observed.Defects in priming are also observed in solid cancers with a low mutational burden and the use of CD40 agonists is able to potentiate T cell responses to solid tumors and might be used in combination to improve ICIs therapy [79,80]. Co-stimulation and co-inhibition is another process in T cell activation which is often impaired in cancer.As previously discussed, the expression of inhibitory receptors (immune checkpoints, \"signal 4\") on T cells represents a physiological mechanism aimed at mitigating tissue damage and autoimmunity [81] (Figure 2).This mechanism is exploited by tumors: cancers suppress immune responses by expressing ligands for negative regulatory receptors on T cells, such as CTLA-4, PD-1, LAG-3 and TIM-3.CTLA-4 signaling is more involved in preventing the initiation of a T cell response in the lymph nodes, while PD-1 serves to limit T cell activity in the TME [82].After TCR engagement, during the priming phase, CTLA-4 is upregulated to attenuate T cell responses and limit the expansion of autoreactive T cells.After the initial success in melanoma, anti-CTLA-4 antibodies like ipilimumab were tested in solid tumors such as breast cancer with limited efficacy [83], though it has been recently approved for the treatment of lung cancer in combination with the anti-PD1 antibody nivolumab in advanced NSCLC [84]. Another molecular mechanism subverted by cancer cells is the very last step of the cancer-immunity cycle, namely the killing of tumor cells by T cells.A functional CRISPR/Cas9-based genome-wide knockout screen performed in B-ALL identified the death receptor signaling pathway as a resistance mechanism to CD19 CAR T-induced cell death [85], suggesting that cancer can also escape CAR T cell killing.Lacking the pro-apoptotic signaling molecules BH3-interacting domain death agonist (BID) or fasassociated protein with death domain (FADD), B-ALL cells were resistant to CD19 CAR T cytotoxicity, leading to disease progression in mice, persistence of tumor cells, which exacerbated T cell dysfunction.Those findings were validated ex vivo after CD19 CAR T cell treatment in B-ALL patients [85]: expression of death receptor pathway genes in pre-treatment samples correlated with CAR T cell expansion and persistence, as well as patient overall survival. By-stander cells in TME can be subverted by cancer cells to suppress T cell function: it is the case of immunosuppressive M2 macrophages, Tregs and MDSCs.The increased fraction of Tregs identified in both solid and hematopoietic cancers inhibits anti-tumor immunity by various mechanisms, such as the constitutive expression of CTLA-4, inhibiting CD80/86 co-stimulatory signals on APC (Figure 2) or direct killing of effector T cells via Fas-FasL [86].MDSCs are also increased in both solid (e.g., breast cancer and melanoma) and hematopoietic malignancies (e.g., MM and CLL) [87][88][89][90][91].They have protumorigenic functions in the TME via multiple mechanisms [92]: induction of immunosuppressive cells, such as Tregs [93] or differentiation of macrophages into the M2 phenotype, blocking of lymphocyte homing via downregulation of cell adhesion molecules on T cells, expression of checkpoints such as PD-L1 [94].M2 macrophages are often expressing PD-L1 or ligands for CTLA-4 [95], providing T cells with the inhibitory \"signal 4\" dampening activation. For reason of limited space, we indicated excellent reviews on bystander cells, extensively discussing the role of Tregs [86], macrophages [95] and MDSCs [87,91,92].Their immunosuppressive role is gaining importance as it might turn out to be one of the main reasons for immunotherapy failure.Another mechanism by which by-stander cells contribute to the generation of an immunosuppressive and protumorigenic milieu is the secretion of immunosuppressive cytokines [96] and other soluble signals, which will be discussed in the next section. ", "section_name": "The Immunosuppressive Microenvironment: Physical Interactions", "section_num": "4." }, { "section_content": "In the context of the cancer-immunity cycle, soluble signals such as cytokines represent signal 3 to balance TCR signaling either to a potent immune response (IL-2, IL-17) or a tolerogenic state (IL-10, IL-6), (Figure 1).Other than cytokines, soluble signals that mediate cell-cell communication in the TME are also represented by exosomes [97,98] and (immunomodulatory) metabolites. Inflammatory cytokines such as TNF-α, IL-6, TGF-β, and IL-10, have been shown to participate in cancer initiation and progression [99], depending on the balance of pro-and anti-inflammatory cytokines and their relative abundance.Besides cancer cells, immune cells, including M2 macrophages, MDSCs and Tregs, and stromal cells, such as fibroblasts and endothelial cells, synthesize them to regulate proliferation, cell survival, differentiation, immune cell activation, cell migration, and death [96].The most known cytokines to induce T cell dysfunction are IL-10, IL-6 and TNF-α, while TGF-β role in cancer is mostly related to epithelial-to-mesenchymal transition (EMT), invasion and metastasis in melanoma [100], breast [101,102] and lung cancer [103]. IL-10 is a potent anti-inflammatory cytokine, secreted by cancer and immune cells [104,105].IL-10 acts on an autocrine manner on naïve T cells promoting their differentiation into Tregs and on effector T cells limiting their proliferation [106].Moreover, IL-10 suppresses APC proinflammatory responses: due to its immunosuppressive effect on DCs and macrophages, IL-10 can dampen antigen presentation allowing tumor cells to evade immune surveillance [107] (Figure 1).IL-10 plays a role in inducing suppression of anti-tumor immunity in CLL [108], contributing to the state of cancer-induced T cell dysfunction. Another pro-inflammatory cytokine with a typical pro-tumorigenic and immunosuppressive effect is IL-6.Elevated IL-6 levels have been detected in serum of patients with systemic cancers.IL-6 has a role in multiple myeloma development, where malignant cells induces senescence in bone marrow stromal cells [39] and cytotoxic clonal T cells [40].Present at high levels in MM microenvironment, IL-6 plays a crucial role in preventing apoptosis in favor of senescence [109]. Cytokines and soluble factors in general play a relevant role in shaping the immune composition in solid cancer as well.To give an example, CD8+ T cells infiltrating the lung TME and the pleural effusion are dysfunctional: they are unresponsive or poorly responsive to any T cell activating stimulus and functionally impaired [110].Surprisingly, TILs or T cells in the pleural effusion show an elevated ratio of memory CD8+ T cells, attracted in a subset-specific manner by chemokines such as CCL21, CCL5, CCL2 [111] (Figure 1).Effector CD8+ T cells are less represented in the TIL population.Two mechanisms could lead to the absence of this subset, namely cell death or tumor cells/TME blocking the differentiation process from memory cells to terminally differentiated effector CD8+ T cells.Immunosuppressive factors in the microenvironment can prevent such differentiation.Lung cancer cell lines secrete IL-6, IL-10 and TGFβ [112,113], which are also found in pleural effusions [103].IL-8 is also secreted by lung cancer cells: this factor is a potent chemoattractant (Figure 1) and recent studies indicated that, besides being involved in EMT of epithelial cancer cells, paracrine signaling by tumor derived IL-8 promotes recruiting of MDSCs into the tumor [114], dampening anti-tumor responses. By-stander cells in the TME are also educated by cancer cells to secrete soluble factors negatively affecting T cell immunity.Besides the above mentioned cytokines, MDSCs and M2 macrophages release nitric oxide (NO) and reactive oxygen species (ROS) [91] that impair T cell activation, via disruption of TCR and eventually induce apoptosis of T lymphocytes [115].Another mechanism adopted by MDSCs is the expression of the enzyme indoleamine 2,3-dioxygenase (IDO), involved in the catabolism of the metabolite tryptophan that has profound immunosuppressive effects on T cells. The tumor microenvironment is poor in nutrients due to the high metabolic demand of malignant cells; solid cancers are characterized by gradients of nutrients, hypoxia and by-products which can act as immunosuppressive metabolites.To give some examples, adenosine is known to affect priming [116] and function [117] of T lymphocytes; kynurenine mediates exhaustion of T cells in melanoma [118], lactate can impair effector functions such as IFNγ production [119] (Figure 1). We will expand in the next section on why metabolism is important in cancer immunity (dys)function. ", "section_name": "The Immunosuppressive Microenvironment: Soluble Signals", "section_num": "5." }, { "section_content": "Metabolic reprogramming is inherently linked to T cell development, activation, function, differentiation, and survival [120,121].After T cell activation, increased demands for energy (ATP), as well as biosynthetic precursors for proliferation, are met by rewiring cellular metabolism.Upon stimulation, co-stimulatory molecules such as CD28 or 4-1BB allow previously quiescent T cells to augment their glycolytic capacity.Downstream to TCR engagement and co-stimulation, activation of PI3K, Akt, and mTOR triggers the switch to anabolic metabolism via the transcription factors Myc and hypoxia-inducible factor 1 (HIF1) [122,123].Interestingly, inhibitory receptors such as PD-1 and CTLA-4 partially act by reducing activation-induced glucose uptake [122,124].Subsequent to glycolysis, oxidative metabolism is augmented, together with increase in the mitochondrial mass (Figure 3).Respiration is therefore also essential for proliferating T cells.adenosine is known to affect priming [116] and function [117] of T lymphocytes; kynurenine mediates exhaustion of T cells in melanoma [118], lactate can impair effector functions such as IFNγ production [119] (Figure 1).We will expand in the next section on why metabolism is important in cancer immunity (dys)function. ", "section_name": "Immunotherapy Efficacy Is Linked to T Cell Fitness-The Survival of the Fittest", "section_num": "6." }, { "section_content": "Metabolic reprogramming is inherently linked to T cell development, activation, function, differentiation, and survival [120,121].After T cell activation, increased demands for energy (ATP), as well as biosynthetic precursors for proliferation, are met by rewiring cellular metabolism.Upon stimulation, co-stimulatory molecules such as CD28 or 4-1BB allow previously quiescent T cells to augment their glycolytic capacity.Downstream to TCR engagement and co-stimulation, activation of PI3K, Akt, and mTOR triggers the switch to anabolic metabolism via the transcription factors Myc and hypoxia-inducible factor 1 (HIF1) [122,123].Interestingly, inhibitory receptors such as PD-1 and CTLA-4 partially act by reducing activation-induced glucose uptake [122,124].Subsequent to glycolysis, oxidative metabolism is augmented, together with increase in the mitochondrial mass (Figure 3).Respiration is therefore also essential for proliferating T cells.T lymphocytes exhibit distinct energy demands according to their differentiation status.Naïve, quiescent T cells predominantly use a resting metabolism relying on oxidative phosphorylation (OXPHOS) to produce ATP: the majority of pyruvate derived from glucose and other fuels (not shown) enters the mitochondria.Early after activation, effector T cells switch to a more glycolytic state to sustain cell growth and proliferation: glucose is mainly fermented into lactate; mitochondria appear punctate.After memory formation, T cells acquire a quiescent metabolic state predominantly fueled by β-oxidation of fatty acids (FAO) that ensures longevity after priming.Memory T cells are also characterized by a highly fused mitochondrial network.Note: there is an ongoing debate whether memory T cells derive from effector T cells, and/or directly from naive precursors after T cell activation.For clarity we have represented the first option, while not excluding the latter. Glycolysis is required to obtain energy, biomass and precursors essential to initiate the differentiation program towards dividing cells and is also crucial for the production of effector molecules like IFN-γ, IL-17 and Granzyme B in T cells [125][126][127][128]. Rewiring of cellular metabolism is also a well-known feature of cancer cells [1], which are adopting a so-called \"aerobic glycolysis\", otherwise referred to as Warburg effect.Even in presence of oxygen, cancer cells prefer the fermentation of glucose into lactate over oxidative metabolism.This choice does not depend on mitochondrial defects nor on impaired oxidative phosphorylation, which is instead required for cancer progression T lymphocytes exhibit distinct energy demands according to their differentiation status.Naïve, quiescent T cells predominantly use a resting metabolism relying on oxidative phosphorylation (OXPHOS) to produce ATP: the majority of pyruvate derived from glucose and other fuels (not shown) enters the mitochondria.Early after activation, effector T cells switch to a more glycolytic state to sustain cell growth and proliferation: glucose is mainly fermented into lactate; mitochondria appear punctate.After memory formation, T cells acquire a quiescent metabolic state predominantly fueled by β-oxidation of fatty acids (FAO) that ensures longevity after priming.Memory T cells are also characterized by a highly fused mitochondrial network.Note: there is an ongoing debate whether memory T cells derive from effector T cells, and/or directly from naive precursors after T cell activation.For clarity we have represented the first option, while not excluding the latter. Glycolysis is required to obtain energy, biomass and precursors essential to initiate the differentiation program towards dividing cells and is also crucial for the production of effector molecules like IFN-γ, IL-17 and Granzyme B in T cells [125][126][127][128]. Rewiring of cellular metabolism is also a well-known feature of cancer cells [1], which are adopting a so-called \"aerobic glycolysis\", otherwise referred to as Warburg effect. Even in presence of oxygen, cancer cells prefer the fermentation of glucose into lactate over oxidative metabolism.This choice does not depend on mitochondrial defects nor on impaired oxidative phosphorylation, which is instead required for cancer progression [129].Glycolysis has various benefits [130] for rapidly proliferating cancer cells, counteracting the selective pressures imposed by TME and, perhaps, by the immune system.At the same time, cancer cells maintain a high metabolic plasticity [131] in order to cope with fluctuations in nutrients and oxygen levels during cancer progression, resulting in the survival of the fittest.Additionally, in multiple tumors, including breast and colon cancer, a fraction of quiescent cells presumably maintaining an oxidative phosphorylation (OXPHOS)-based metabolism [132] is responsible for resistance and relapse to therapy, as well as immune escape [133]. The Warburg effect indeed describes tumor metabolism only partially [134], and the high metabolic heterogeneity within tumors is achieved through a complex network of interactions between different compartments, such as cancer cells, by-stander cells and stromal cells.This metabolic coupling allows the transfer of metabolites from stromal cells to support cancer cells in their response to fluctuations of nutrients [135].Interestingly, a recent model, namely the reverse Warburg effect, describes a two-compartment interplay in which stromal and by-stander cells are induced by cancer to assume a glycolytic phenotype, whose catabolic products (lactate, pyruvate, ketone bodies) are then used by cancer cells for mitochondrial OXPHOS.It has been recently proposed that a more complex twodimensional model including stroma and cancer metabolism should be taken into account when assessing immunotherapy efficacy [136]. Studies correlating cancer metabolism and immune dysfunction are flourishing.Interestingly, cytokine secretion by tumor cells is regulated by external stimuli and stressors such as glucose deprivation, as shown in a model of lung cancer [137] and this might represent an additional mechanism of immune regulation in solid tumors. As shown by experiments conducted in murine models of melanoma and sarcoma, in solid tumors the mechanisms underlying T cell metabolic dysfunction can be rooted in metabolic competition for glucose [119,138], or in the immunosuppressive effect of lactate [139] or other by-products of cancer cells, such as the previously mentioned tryptophan or kynurenine (Figure 1). In addition to melanoma and sarcoma murine models, in CLL patients T cells have defects in activation, accompanied by the inability to properly upregulate glucose uptake and glycolytic metabolism [140].Immune suppression by glucose competition has apparently been excluded in CLL, as well as a role for lactate in immune suppression [141].CLL seems indeed an exception to the Warburg rule: CLL cells take advantage from catabolism of fatty acids, while maintaining a high metabolic plasticity to counteract selective pressures in TME [142].T cells from B-ALL patient showed a similar deficiency in activation and glycolytic metabolism upon stimulation [140].Those defects did not impede the generation of functional and highly persistent CAR T cells which lead to enormous successes in B-ALL treatment, while the efficacy is still very limited in CLL.It might be speculated that T cell dysfunction in CLL is exacerbated by chronic stimulation.In a recent report, it has been shown in an in vitro model that persistent antigenic stimulation leads to a loss of mitochondrial function [143], due to the induction of mitochondrial oxidative stress that limits ATP production via OXPHOS.Other studies are underscoring the importance of mitochondria for T cell function [144], such as a recent report showing that T cell exhaustion is reinforced by deregulated mitochondrial dynamics (fission and fusion) regulating the turnover of those organelles [145].Interestingly, the ultrastructure of mitochondria also differs in memory versus effector T cells [146].The latter are characterized by punctate mitochondria, while memory T cells maintain a highly fused network of these organelles (Figure 2). In addition, metabolic plasticity also regulates differentiation and skewing of T cells, with terminally exhausted effector cells showing a glycolytic metabolism, in contrast to fatty acid β-oxidation characterizing memory subsets [147], (Figure 3).Thus, the hypoxic, acidic, scarce-in-nutrients tumor microenvironment has a negative impact on T cell differentiation.The hypoxic conditions in the TME, for example, prevent the acquisition of the memory T cell phenotype, which relies heavily on oxygen [27].It is widely reported that the memory phenotype increases T cell survival, proliferation and prolonged presence of TILs and CAR T cells at tumor sites [143,148].Patients with prevalence of memory T cells at the tumor site have increased survival [149] and the state of differentiation exhibited by the apheresed T cells affects the efficacy and persistence of the infusion CAR T cell product [150]. In the last decade we witnessed the rise of the immunometabolism field and studies on the topic have increased exponentially.Reports describing the alterations in T cell metabolism can have profound implications in the optimization of immunotherapeutic strategies.In view of those findings, researchers are making efforts in the direction of manipulating the metabolism of either TILs or CAR T cells to more efficiently utilize nutrients, with the aim of maximizing their performance and prolong their survival, eventually improving the efficacy of cancer immunotherapy.The design of CAR constructs can be improved by 'armoring' T cells with enhanced metabolic functions.On the other hand, this knowledge could also be beneficial in improving ICIs therapy when combined with drugs targeting and fine-tuning metabolism as adjuvant therapy. ", "section_name": "Immunotherapy Efficacy Is Linked to T Cell Fitness-The Survival of the Fittest", "section_num": "6." }, { "section_content": "In summary, hematopoietic and solid cancers are showing different defect in central versus peripheral tolerance, with B-cell lineage malignancies displaying a more unique defect in TCR selection and priming, and solid cancers presenting adequate immune response initiation which is then dampened at the tumor site. Many trials are testing combinatorial approaches of immunotherapy with other treatments.A strategy could be optimizing the schedule of immunotherapy administration, for example after surgery to remove tumor cells inducing T cell dysfunction, taking care not to exacerbate it via chemotherapy.An early use of immunotherapy to treat patients with early-stage cancers could lead to an improved efficacy.To date, immunotherapy has primarily been used in patients with advanced stage or relapsed cancers.However, multiple trials are addressing the benefit of the use of immunotherapy in early-stage tumors.Hopefully, those ongoing trials will reveal even better results than those obtained in advanced settings. Studies reporting the importance of metabolism in cancer immunotherapy are flourishing, but still a complete overview of metabolic interactions between malignant and immune cells in the various cancer types is lacking.A deeper investigation of immunometabolism should indeed lead to a profound improvement of immunotherapeutic strategies: ICIs approaches could benefit from the administration of adjuvant therapies targeting metabolism and nutrient utilization, and CAR T cell persistence could be boosted by armoring the chimeric TCR with genes involved in the regulation of cellular metabolism. ", "section_name": "Conclusions", "section_num": "7." } ]	[ { "section_content": "We thank our colleagues Anne W.J. Martens, Department of Experimental Immunology, Cancer Center Amsterdam, Amsterdam Infection & Immunity Institute, and Lymphoma and Myeloma Center Amsterdam, Amsterdam UMC, University of Amsterdam, The Netherlands, Ann Zeuner, Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy, Joris D. Veltman, Department of Respiratory Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands and Rosalie M. Luiten, Netherlands Institute for Pigment Disorders, Department of Dermatology, Amsterdam Infection and Immunity ", "section_name": "Acknowledgments:", "section_num": null }, { "section_content": "Funding: This work was supported by the EU's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement 766214 (META-CAN).CMP's lab is funded by the CERCA Program/Generalitat de Catalunya and by Ministerio de Ciencia, Innovación y Ministerio de Ciencia e Innovación, part of Agencia Estatal de Investigación (AEI), through the Generación de Conocimiento grant, number PID2019-107213GB-I00/DOI: 10.13039/501100011033. ", "section_name": "", "section_num": "" }, { "section_content": "Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands, for reading the manuscript, comments and criticisms. The authors declare no conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null }, { "section_content": "Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands, for reading the manuscript, comments and criticisms. ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare no conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.3390/biomedicines12092009	Increased Myocardial MAO-A, Atrogin-1, and IL-1β Expression in Transgenic Mice with Pancreatic Carcinoma—Benefit of MAO-A Inhibition for Cardiac Cachexia	<jats:p>Cancer cachexia (CC) continues to challenge clinicians by massively impairing patients’ prognosis, mobility, and quality of life through skeletal muscle wasting. CC also includes cardiac cachexia as characterized by atrophy, compromised metabolism, innervation and function of the myocardium through factors awaiting clarification for therapeutic targeting. Because monoamine oxidase-A (MAO-A) is a myocardial source of H2O2 and implicated in myofibrillar protein catabolism and heart failure, we presently studied myocardial MAO-A expression, inflammatory cells, and capillarization together with transcripts of pro-inflammatory, -angiogenic, -apoptotic, and -proteolytic signals (by qRT-PCR) in a 3x-transgenic (LSL-KrasG12D/+; LSL-TrP53R172H/+; Pdx1-Cre) mouse model of orthotopic pancreatic ductal adenoarcinoma (PDAC) compared to wild-type (WT) mice. Moreover, we evaluated the effect of MAO-A inhibition by application of harmine hydrochloride (HH, 8 weeks, i.p., no sham control) on PDAC-related myocardial alterations. Myocardial MAO-A protein content was significantly increased (1.69-fold) in PDAC compared to WT mice. PDAC was associated with an increased percentage of atrogin-1+ (p < 0.001), IL-1β+ (p < 0.01), COX2+ (p < 0.001), and CD68+ (p > 0.05) cells and enhanced transcripts of pro-inflammatory IL-1β (2.47-fold), COX2 (1.53-fold), TNF (1.87-fold), and SOCS3 (1.64-fold). Moreover, PDAC was associated with a reduction in capillary density (−17%, p < 0.05) and transcripts of KDR (0.46-fold) but not of VEGFA, Notch1, or Notch3. Importantly, HH treatment largely reversed the PDAC-related increases in atrogin-1+, IL-1β+, and TNF+ cell fraction as well as in COX2, IL-1β, TNF, and SOCS3 transcripts, whereas capillary density and KDR transcripts failed to improve. In mice with PDAC, increased myocardial pro-atrophic/-inflammatory signals are attributable to increased expression of MAO-A, because they are significantly improved with MAO-A inhibition as a potential novel therapeutic option. The PDAC-related loss in myocardial capillary density may be due to other mechanisms awaiting evaluation with consideration of cardiomyocyte size, cardiac function and physical activity.</jats:p>	[ { "section_content": "Cancer cachexia (CC) is experienced by a majority of cancer patients and by definition entails skeletal muscle wasting, which massively compromises the patients' physical performance, pulmonary function, quality of life, tolerance to chemotherapy, and overall prognosis [1][2][3][4][5].The still largely untreatable CC syndrome is highly prevalent in patients with gastrointestinal carcinoma, with the most rapid and devastating development observed with pancreatic ductal adenocarcinoma (PDAC) [1,[5][6][7].The phenotype of skeletal muscle wasting and its underlying pro-oxidative/-inflammatory and (neuro)endocrine mechanisms leading to net-proteolysis, apoptosis, and impaired regeneration via NF-κB-, STAT3-, myostatin-, insulin-Akt, or other pathways have been extensively studied within the last two decades [5,[8][9][10][11][12][13][14][15][16].However, relevant pro-cachectic triggers of myofibrillar catabolism may not spare the heart.Indeed, cardiac cachexia, i.e., decreased myocardial mass and function, has now been recognized as a largely understudied factor within CC [17,18] that independently impairs cancer patients' prognosis [19], contributes to CCrelated autonomic dysregulation [20], and is mutually interrelated to inactivity/skeletal muscle wasting [21].In addition to myocardial atrophy and dysfunction, cardiac cachexia includes functional, structural, or metabolic remodeling, denervation, and mitochondrial dysfunction among other factors [18,22,23].At present, the underlying mechanisms remain to be identified for effective therapeutic targeting [18] although they might not be fundamentally different from established factors driving skeletal muscle wasting [5,8,11]. The mitochondrial flavoenzyme monoamine oxidase-A (MAO-A) catalyzes the degradation of monoamine neurotransmitters and dietary amines, thereby generating H 2 O 2 , ammonium, and aldehyde [24][25][26].Together with NADPH oxidases, xanthine oxidases, nitric oxide synthases, and the mitochondrial respiratory chain, MAO-A is an important source of ROS that is physiologically expressed in the myocardium [27][28][29][30].High MAO-A expression or catecholamine-related activation and related oxidative stress have been strongly implicated in heart failure or experimental pressure overload, myocardial hypoxia-reoxygenation injury, diabetes-associated myocardial dysfunction, and cardiac aging [27,29,[31][32][33].For example, inhibition of MAO-A was shown to improve myocardial recovery from acute volume overload by aortal-caval fistula in rats [34] or from cardiac arrest [35] and to protect against catecholamine-induced arrhythmias [36]. Though largely understudied in the context of CC, MAO-A has been suggested to be a potential driver of and therapeutic target in skeletal muscle wasting.In an in-vitro model of steroid-induced skeletal muscle wasting, MAO-A was found to be massively upregulated [37].Moreover, we have most recently shown that transgenic PDAC-bearing cachectic mice in comparison to WT mice revealed increased MAO-A protein expression in hindlimb skeletal muscle undergoing fiber atrophy [9,38].Interestingly, selective reversible MAO-A inhibition with harmine hydrochloride (HH) significantly decreased pro-atrophic (atrogin-1, MuRF1) and pro-inflammatory (IL-1β, SOCS3) transcripts in hindlimb muscles of mice in this CC model.However, such desirable therapeutic effects failed to translate into reversal of muscle wasting, likely due to damage of neuromuscular junctions in fast-twitch skeletal muscle through the rather high HH dose of 30 mg kg -1 day -1 for 8 weeks [38]. Using this triple transgenic mouse model of PDAC-related CC, we presently aimed to study the role MAO-A in cardiac cachexia, hypothesizing that MAO-A inhibition by HH, i.e., lowering H 2 O 2 generation, would attenuate pro-oxidative/-inflammatory/-atrophic alterations of the myocardium without the specific neuromuscular adverse effects observed in skeletal muscle [38]. High MAO-A expression/activity may also interfere conditionally and unpredictably with VEGF expression and angiogenesis, at least in cancer mouse models.MAO-A may contribute to HIF-1α stabilization, promoting VEGF expression, ROS production, tumorigenesis, and metastasis [39], whereas, under conditions of hypoxia and IL-6 receptor activation in breast cancer cells, enhanced VEGF expression is associated with MAO-A suppression [40].Given the relevance of capillarization for cardiac function, not assessed so far with CC, we also determined myocardial capillary density with and without HH treatment in PDAC and WT mice. The present study reports, for the first time, a significant increase in myocardial MAO-A expression with PDAC (-related cachexia) in combination with increases in the percentage of atrogin+-, IL-1β+-, and COX2+-cells as well as in related transcripts of IL-1β, COX2, TNF, and SOCS3 genes.As evidence for MAO-A as a causal pro-atrophic/-inflammatory driver and future therapeutic target, our data show that MAO-inhibition by HH mostly reversed almost all of these pro-cachectic/-inflammatory impairments. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "", "section_name": "Material and Methods", "section_num": "2." }, { "section_content": "The presently studied triple transgenic mouse model of PDAC-introduced and provided by Hingorani et al.-is based on the clinically highly PDAC-relevant point mutations of transformation-related protein 53 (Trp53, R172H/+: arginine/histidine substitution in codon 172) and Kirsten rat sarcoma (Kras, G12D/+: glycine/aspartate substitution in codon 12) [41,42] and develops muscle wasting as described [9,38].Activation of the combined mutants upon crossbreeding with the pancreas specific Pdx-1-Cre promoter leads to orthotopic PDAC development via progressive inflammation-driven precancerous lesions (pancreatic intraepithelial neoplasia, PanIN grades 1-3).WT mice with a BL/6 background served as controls.All mice were maintained by the Biomedical Research Centre of the University of Marburg under conditions of food and water supply ad libitum as well as a periodic day-night cycle of 12 h. Out of 42 mice in total (23 WT and 19 PDAC mice), subgroups of 11 WT and 9 PDAC mice were treated with HH (30 mg/kg/day, i.p.) for two months, starting at the age of three months.After the treatment, mice were sacrificed, their hearts removed, and non-apical/basal samples of the left myocardium for qT-PCR or Western blotting as well as transversal sections at maximal circumference for cryo-sections (embedded in Tissue-Tek™, Sakura Finetek, Stauffen, Germany, and submersed in precooled isopentane) were snap-frozen using liquid nitrogen.The histopathological diagnosis of PDAC or normal pancreas was independently assessed by two experienced investigators.The study was approved by the Regional Commission Giessen (MR 20/11-Nr.70/2009)and conducted according to the regulations for animal experiments at the Philipps University, Marburg. ", "section_name": "Transgenic Mouse Model of PDAC", "section_num": "2.1." }, { "section_content": "Ventricular left myocardial samples were lysed in radioimmunoprecipitation assay (RIPA) buffer at pH 7.5 (Cell Signaling Technology Europa, Leiden, The Netherlands) with protease/phosphatase inhibitors (Cell Signaling Technology, Boston, MA, USA) and their total protein concentrations determined by means of the Pierce BCA (bicinchoninic acid) assay (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions.Proteins were loaded onto pre-cast polyacrylamide NuPAGE ® 4-12% Bis-Tris gels (Life Technologies GmbH, Darmstadt, Germany).Following SDS-PAGE, proteins were transferred onto 0.45 µm nitrocellulose membranes (Millipore; Billerica, MA, USA).Membranes were stained for MAO-A (rabbit monoclonal anti-MAO-A antibody, ab126751, Abcam, Cambridge, UK) and for α-tubulin (1:2000, ab4074, Abcam, Cambridge, UK) by incubation with the respective immunoreactive primary antibodies overnight at 4 • C in blocking buffer.After washing with TBS 0.1% Tween 20, membranes were incubated with ECL anti-rabbit IgG, horseradish peroxidase (HRP) secondary antibody (MA9340, GE Healthcare, Amersham, UK).The peroxidase reaction was visualized with ImmobilonTM Western (HRP) substrate (Merck Chemicals GmbH.Darmstadt, Germany).The intensity of WB bands was quantified using ImageJ/Fiji software (version 1.54f) from the National Institutes of Health (Bethesda, MD, USA). ", "section_name": "Western Blotting of MAO-A Expression", "section_num": "2.2." }, { "section_content": "Transversal or longitudinal cryo-sections of the ventricular left myocardium were fixed by 5% paraformaldehyde (PFA) for 10 min, blocked with 1% normal swine serum (NSS) at 37 • C, and incubated with primary rabbit anti-mouse polyclonal antibodies against atrogin-1 (FBXO32, MAFbx, 1:200, ab74023, Abcam, Cambridge, UK), against IL-1β (1:50, ab9722, Abcam, Cambridge, UK), TNF (1:100, ab6671, Abcam, Cambridge, UK) or against COX2 (1:200, ab15191, Abcam, Cambridge, UK), and primary rat anti-mouse primary monoclonal antibodies against CD68 (1:50, MCA1957, AbD Serotec, Kidlington, UK).Their respective detection was enabled by anti-rabbit IgG (1:200, ZRH1158, Linaris, Biological Products, Dossenheim, Germany) or anti-rat IgG (1:200, STAR72, AbD Serotec, Kidlington, UK) secondary antibodies that were conjugated with horseradish peroxidase (HRP) for catalyzation of oxidation with H 2 O 2 of 3,3 ′ -diaminobenzidine (DAB) as a chromogenic substrate.Nuclei were counterstained with Mayer's hematoxylin.Using digital images (200×) obtained by the Zeiss Axio Imager M2 microscope combined with Axio-Cam HRc/AxioVision, the total number of immunohistochemically positive cell and counterstained nuclei were analyzed in three rectangular areas (0.145 mm 2 ) of both transversal (n ≥ 3) and longitudinal (n ≥ 3, except for atrogin-1 staining) representative tissue sections using ImageJ processing package Fiji software 1.54f.The density of immunolocalized cells was assessed in terms of percentage (%) of counterstained hematoxylin+ nuclei in total (to enable control for possible changes in cardiomyocyte size). ", "section_name": "Immunolocalization of Atrogin-1+, IL-1β+, TNF+, COX2+, and CD68+ Cells", "section_num": "2.3." }, { "section_content": "Ventricular left myocardial capillary density (CD) was determined by histochemical visualization of capillaries in 7-µm cryo-cross-sections fixed in 4% paraformaldehyde/ phosphate-buffered saline (PFA/PBS) for 10 min at room temperature as previously described [9,38].Briefly, after blocking endogenous peroxidases with hydrogen peroxide (0.05% in PBS; pH 7.4), the cross-sections were incubated for 30 min at 37 • C with 40 µg/mL horseradish peroxidase-conjugated Isolectin B4 of Bandeiraea simplicifolia (BSI-B4, Sigma-Aldrich Co. LLC, St. Louis, MO, USA) in PBS or with D-galactose for negative control staining, and 3-3 ′ -diaminobenzidine (DAB, Merck-Sigma-Aldrich Co. LLC, St. Louis, MO, USA) was used for detection of capillaries.Nuclei were counterstained with Mayer's hematoxylin (Carl Roth GmbH & Co. KG, Karlsruhe, Germany).Digital images (200×) were obtained by the Zeiss Axio Imager M2 microscope (Carl Zeiss AG; Oberkochen, Germany) combined with Axio-Cam HRc/AxioVision software Release 4.8.2 (Carl Zeiss GmbH, Oberkochen, Germany).Three separate rectangular cross-sectional areas (0.145 mm 2 ) were used to assess CD in terms of counts of capillaries (diameter ≤ 7.5 µm) per area using ImageJ/Fiji software (National Institute of Health, Bethesda, MD, USA). ", "section_name": "Myocardial Capillary Density", "section_num": "2.4." }, { "section_content": "RNA extraction was carried out using peqGOLDTriFast™ (VWR International GmbH, Darmstadt, Germany) according to the manufacturer's instructions.RNA concentration was measured via optical density (OD, 260 nm) as well as its purity assessed (OD260 nm/OD280 nm, with a ratio of 1.8 to 2.0 accepted as pure) using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).A high RNA integrity (RIN, between 8 and 10) for reverse transcription (RT) was confirmed by means of an RNA 6000 NanoChip kit on an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA).An aliquot of total RNA was treated with one unit DNAse (Thermo Fisher Scientific Inc.) for 30 min at 37 • C and RT performed for 1 h at 42 • C using oligo primer (dT) 12-18 (Agilent Technologies Inc.), 20 units of the reverse transcriptase included in the Affinity Script multiple-temperature cDNA synthesis kit (Agilent Technologies Inc.), 24 units of Ribo Lock™ RNAse inhibitor (Thermo Fisher Scientific Inc.), and 4 mM dNTP mix (Agilent Technologies Inc.).The cDNA was used for qRT-PCR using the QuantiTectprimer assays (Table 1) (Qiagen N.V., Venlo, Netherlands) combined with Takyon™ Low Rox Probe Master-Mix dTTP Blue (Eurogentec, Seraing, Belgium) or Agilent™ Brilliant III Ultra-Fast SYBR ® Green QPCR Master-Mix (Agilent Technologies Inc.).The thermal profile consisted of 3 min at 95 • C followed by 45 cycles at 95 • C for 10 s and 60 • C for 20 s.The qPCR and data analyses were performed using the Stratagene Mx3005P™ qPCR System (Agilent Technologies, Inc.).The relative amount within each sample was calculated by linear regression analysis from their respective standard curves generated from a cDNA pool.The specificity of the amplified product was confirmed by the melting curve analysis (55-95 • C).The myocardial expression of RPLP0 was selected out of potential candidate genes, including β-actin, GAPDH, etc., as a housekeeping gene using Normfinder software version 20 as described [38] (Table 1).All Primers were purchased from Qiagen N.V., Venlo, the Netherlands. ", "section_name": "Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)", "section_num": "2.5." }, { "section_content": "Data are presented as mean ± standard error of the mean (SEM) for the groups WT, WT-HH, CA, and CA-HH.To detect a significant and independent impact of PDAC and/or of HH treatment, a two-factorial ANOVA (with or without age or sex as covariates) was applied to the total study population and p-values presented for the effect of PDAC (CA and CA-HH group) vs. WT (WT and WT-HH groups) and/or for the effect of HH (WT-HH and CA-HH group) vs. untreated mice (WT and CA group) as well as for the interaction of both these factors in tables and figure legends.Significant differences between two groups were assessed (posthoc) by the Student's t-test or, in case of no normal distribution, by the Mann-Whitney U test, and indicated within the graph by the symbol * (for PDAC effects) or the symbol # (for HH effects).A p < 0.05 was considered to be statistically significant.Given the explorative (hypothesis-generating) nature of the study, no Bonferroni correction was applied.All statistical procedures were performed by SPSS (version 27, IBM Munich, Munich, Germany). ", "section_name": "Statistical Analyses", "section_num": "2.6." }, { "section_content": "", "section_name": "Results", "section_num": "3." }, { "section_content": "As presented in Table 2, the CA group (with histopathological confirmation of PDAC) showed no significant weight loss as compared to the WT group (control with normal pancreatic histology).Obviously, the significant PDAC-related skeletal muscle wasting, as recently reported in this mouse model [9,38], was not reflected by a significant weight loss, likely due to variable tumor, metastasis or fat mass, extent of ascites, or other factors.PDAC-related weight loss did tend to be higher in the male compared to the female CA group as recently reported [9].This is in line with their larger muscle mass in terms of percentage of body weight.Overall, HH treatment had no significant impact on body weight; however, the CA-HH group revealed significantly lower values by 2.4 g.Age did not significantly impact body weight, although the CA group was significantly older than the WT and CA-HH groups by ca.7 and 4 weeks, respectively, while the WT-HH group was older than the WT group by ca. 2 weeks (Table 2). ", "section_name": "Group Characteristics", "section_num": "3.1." }, { "section_content": "Relative MAO-A protein content in the left myocardium (Figure 1) showed a variable, however, overall significant 1.69-fold increase in PDAC-bearing (CA and CA-HH) as compared to WT (WT and WT-HH) mice.ANOVA confirmed a significant effect of PDAC independent of the factors HH treatment, age, or gender, all of which were without significant impact compared to MAO-A protein expression. This PDAC-related increase in protein level was significant, with normalization for both tubulin (Figure 1) and Ponceau staining of total protein abundance.Notably, no parallel PDAC-related increase in myocardial MAO-A (or MAO-B) transcripts was observed (Table 3).This PDAC-related increase in protein level was significant, with normalization fo both tubulin (Figure 1) and Ponceau staining of total protein abundance.Notably, no par allel PDAC-related increase in myocardial MAO-A (or MAO-B) transcripts was observed (Table 3). ", "section_name": "Increased MAO-A Protein Content in PDAC Compared to WT Mice", "section_num": "3.2." }, { "section_content": "The density of atrogin-1+ or other immunolocalized (see below) cells was expressed in terms of percentage of total hematoxylin+ nuclei, which, notably, did not significantly differ in terms of count per area (n/mm 2 ) between the four groups of WT (2037 ± 74), WT-HH (2013 ± 54), CA (1861 ± 109), and CA-HH (2113 ± 77).As a main finding (Figure 2a,b), there was a highly significant more than two-fold PDAC-related increase in percentage of atrogin-1+ cells (atrogin-1+ cells in % of hematoxylin+ cells) in myocardial cross-sections.Immunohistochemical staining shows that the PDAC-related increase in atrogin-1 expression actually occurs within cardiomyocytes (Figure 2b).HH treatment significantly but incompletely reversed the increase in percentage of atrogin-1+ cardiomyocytes to a level that remained significantly above that of the WT mice (control). there was a highly significant more than two-fold PDAC-related increase in percentage of atrogin-1+ cells (atrogin-1+ cells in % of hematoxylin+ cells) in myocardial cross-sections.Immunohistochemical staining shows that the PDAC-related increase in atrogin-1 expression actually occurs within cardiomyocytes (Figure 2b).HH treatment significantly but incompletely reversed the increase in percentage of atrogin-1+ cardiomyocytes to a level that remained significantly above that of the WT mice (control) such that its level remained significantly above the WT group (control) level.In WT mice, HH treatment appeared to have little effect on percentage of atrogin-1+ cardiomyocytes.ANOVA confirmed significant, independent opposing effects of PDAC and of HH, while age and sex had no significant impact.Notably, there was a significant In WT mice, HH treatment appeared to have little effect on percentage of atrogin-1+ cardiomyocytes.ANOVA confirmed significant, independent opposing effects of PDAC and of HH, while age and sex had no significant impact.Notably, there was a significant positive correlation between percentages of atrogin-1+ cardiomyocytes (r = 0.687 p = 0.001) and MAO-A protein contents among the total study population, i.e., irrespective of the (incompletely reversing) effect of HH.In contrast, myocardial atrogin-1 transcript levels were found to be largely unaffected by PDAC or HH treatment in pooled analyses (Table 3). Moreover, the pooled transcript levels of MuRF1, another relevant pro-atrophic ubiquitin E3-ligase, were unaffected by PDAC as well but tended to be higher with HH treatment in the WT and the CA group (Table 3). Regarding apoptosis as another factor of cardiac cachexia, transcripts of Casp3 and BCL-2 were largely unaffected by PDAC and HH treatment except for a <0.50-fold decrease with HH treatment in the CA-HH group compared to the CA or the WT group (Table 3). ", "section_name": "Percentage of Atrogin-1+ Cells Is Increased in PDAC and Reversed by HH-Treatment", "section_num": "3.3." }, { "section_content": "Since IL-1β is a well-established pro-inflammatory trigger of muscle wasting, it was presently considered as another main outcome variable in this context of cardiac cachexia.As presented in Figure 3a,b, percentage of IL-1β+ cells (TNF+ cells in % of hematoxylin cells) significantly increased in PDAC overall, as reflected in CA or CA-HH mice compared to WT or WT-HH mice, respectively.Moreover, this PDAC-related increase in percentage of IL-1β+ cells was significantly reduced with HH-treatment overall and this effect was detectable in comparison to both WT and CA mice, as well.These significantly opposing impacts, PDAC and HH treatment, showed no significant interaction according to ANOVA and were independent of age or sex.In line with these data, IL-1β transcript levels (Figure 3c, Table 3) were found to be almost 2.5-fold higher in CA than in WT mice and even more pronounced in CA-HH than in WT-HH mice, while HH treatment considerably decreased these levels in both PDAC-bearing and WT mice. presently considered as another main outcome variable in this context of cardiac cachexia.As presented in Figure 3a,b, percentage of IL-1β+ cells (TNF+ cells in % of hematoxylin cells) significantly increased in PDAC overall, as reflected in CA or CA-HH mice compared to WT or WT-HH mice, respectively.Moreover, this PDAC-related increase in percentage of IL-1β+ cells was significantly reduced with HH-treatment overall and this effect was detectable in comparison to both WT and CA mice, as well.These significantly opposing impacts, PDAC and HH treatment, showed no significant interaction according to ANOVA and were independent of age or sex.In line with these data, IL-1β transcript levels (Figure 3c, Table 3) were found to be almost 2.5-fold higher in CA than in WT mice and even more pronounced in CA-HH than in WT-HH mice, while HH treatment considerably decreased these levels in both PDAC-bearing and WT mice.As seen in Figure 4a,b, the percentage of TNF+ cells showed a, by trend, 1.45-fold increase in the CA compared to the WT group, which was counteracted by HH treatment, leading to a significant decrease in PDAC-bearing mice.These effects were confirmed in terms of a significant interaction of the factors PDAC and HH treatment according to ANOVA.In line with these data, TNF transcript levels showed a 1.87-fold increase in the CA group vs. the WT group and a strong opposing effect through HH treatment, which decreased TNF transcripts to largely below WT group, i.e., control level (Figure 4c, Table 3).The percentage of TNF+ cells were found to be unrelated to MAO-A protein expression. As presented in Figure 5a,b, moderate but significant PDAC-and HH-related increases in the percentage of CD68+ cells were detected in untreated mice, which was reflected by a significant interaction but no significant impact of factor CA and factor HH within the total study population.The CD68 transcript levels (Figure 5c, Table 3) revealed a similar trend but no relevant change through PDAC or HH treatment. As seen in Figure 4a,b, the percentage of TNF+ cells showed a, by trend, 1.45-fold increase in the CA compared to the WT group, which was counteracted by HH treatment, leading to a significant decrease in PDAC-bearing mice.These effects were confirmed in terms of a significant interaction of the factors PDAC and HH treatment according to ANOVA.In line with these data, TNF transcript levels showed a 1.87-fold increase in the CA group vs. the WT group and a strong opposing effect through HH treatment, which decreased TNF transcripts to largely below WT group, i.e., control level (Figure 4c, Table 3).The percentage of TNF+ cells were found to be unrelated to MAO-A protein expression. As presented in Figure 5a,b, moderate but significant PDAC-and HH-related increases in the percentage of CD68+ cells were detected in untreated mice, which was reflected by a significant interaction but no significant impact of factor CA and factor HH within the total study population.The CD68 transcript levels (Figure 5c, Table 3) revealed a similar trend but no relevant change through PDAC or HH treatment.Figure 6a,b shows a significant increase in the percentage of COX2+ cells with PDAC by ANOVA, which was detectable in the CA and CA-HH groups as compared to the WT and WT-HH groups, respectively.Overall, HH treatment resulted in a significant additional increase in the percentage of COX2+ cells whereby a significant interaction between the factors CA and HH indicated a positive synergism of both effects.The effect of HH treatment on percentage COX2+ cells, however, reached significance in WT but not in CA mice.On the level of transcripts (Figure 6c, Table 3), COX2 showed a 1.53-fold upregula- Figure 6a,b shows a significant increase in the percentage of COX2+ cells with PDAC by ANOVA, which was detectable in the CA and CA-HH groups as compared to the WT and WT-HH groups, respectively.Overall, HH treatment resulted in a significant additional increase in the percentage of COX2+ cells whereby a significant interaction between the factors CA and HH indicated a positive synergism of both effects.The effect of HH treatment on percentage COX2+ cells, however, reached significance in WT but not in CA mice.On the level of transcripts (Figure 6c, Table 3), COX2 showed a 1.53-fold upregulation in the CA group compared to the WT group, which was massively reversed by 0.27-fold through HH treatment.No such effect was observed in the WT-HH compared to the WT group.As shown in Table 3, the transcript level of SOCS3, also implicated in pro-cachectic signaling, was found to be 1.64-fold upregulated in the CA group compared to the WT group, whereas HH treatment decreased these <0.5-fold below WT group (control) level. ", "section_name": "Upregulation of IL-1β, TNF, COX2, CD68, and SOCS with PDAC and Differential Anti-Inflammatory Effect of HH Treatment", "section_num": "3.4." }, { "section_content": "As shown in Figure 7a,b, overall PDAC was associated with a highly significant reduction in capillary density (-15%), which was detectable between the CA and WT groups (-14%) as well as between the CA-HH and WT-HH groups (-16%).In contrast, HH treatment yielded no significant changes in capillary density.Regarding transcript levels of relevant angiogenic signals, VEGFA remained largely unaffected by PDAC or HH treatment (Table 3).However, a 0.48-fold downregulation of its receptor KDR was observed in the CA group compared to the WT group (Figure 7c, Table 3).HH treatment had little effect on KDR in PDAC-bearing or WT mice.As shown in Table 3, the transcript level of SOCS3, also implicated in pro-cachectic signaling, was found to be 1.64-fold upregulated in the CA group compared to the WT group, whereas HH treatment decreased these <0.5-fold below WT group (control) level. ", "section_name": "Effect of PDAC and HH on Myocardial Capillary Density and Angiogenic Signals", "section_num": "3.5." }, { "section_content": "As shown in Figure 7a,b, overall PDAC was associated with a highly significant reduction in capillary density (-15%), which was detectable between the CA and WT groups (-14%) as well as between the CA-HH and WT-HH groups (-16%).In contrast, HH treatment yielded no significant changes in capillary density.Regarding transcript levels of relevant angiogenic signals, VEGFA remained largely unaffected by PDAC or HH treatment (Table 3).However, a 0.48-fold downregulation of its receptor KDR was observed in the CA group compared to the WT group (Figure 7c, Table 3).HH treatment had little effect on KDR in PDAC-bearing or WT mice.The transcripts of Notch1 and Notch3 displayed a trend toward PDAC-related downregulation, which was somewhat reinforced by HH treatment (Table 3). ", "section_name": "Effect of PDAC and HH on Myocardial Capillary Density and Angiogenic Signals", "section_num": "3.5." }, { "section_content": "Cancer cachexia involves catabolic, mostly inflammatory mediators, that not only trigger skeletal muscle wasting but may also promote cardiac cachexia.This includes myocardial atrophy, dysfunction, and related remodeling that critically compromises cancer patients' prognosis and quality of life [17][18][19].The present study aimed to evaluate the role of MAO-A, a novel potential therapeutic target in CC, in the context of cardiac cachexia [38]. Using an established triple transgenic mouse model with orthotopic PDAC [41,42] compared to WT mice, we demonstrate for the first time that PDAC (-related CC) is associated with a significantly higher protein expression of MAO-A in the left myocardium.As a mitochondrial source of H2O2, MAO-A (like MAO-B) contributes to oxidative stress in the myocardium as well as in skeletal muscle [27][28][29][30]33,37,43]. High MAO-A expression or activity is clearly implicated in several cardiac conditions [27,29,31,32] that benefit from MAO-A inhibition [34][35][36].As yet, MAO-A has not been addressed in cardiac cachexia; however, we recently reported, in the presently studied PDAC-related mouse model of CC, that MAO-A protein expression was significantly increased in skeletal (gastrocnemius) muscle.Furthermore, MAO-A inhibition with HH significantly attenuated transcripts of pro-inflammatory IL-1β in the gastrocnemius muscle and of the pro-atrophic E3-ligases atrogin-1 and MuRF1 in the soleus muscle without changing MAO-A protein expression levels [38].Unfortunately, this potentially anti-cachectic effect of HH failed to translate into a reversal of muscle skeletal wasting, likely due to a compromised neuromuscular junction as an adverse effect of HH.Given the principle difference in cardiac innervation, we presently hypothesized that HH treatment could attenuate possible CC-related upregulation of E3 ligases and upstream cytokine triggers responsible for myofibrillar breakdown in myocardium as well as skeletal muscle [17,44].The transcripts of Notch1 and Notch3 displayed a trend toward PDAC-related downregulation, which was somewhat reinforced by HH treatment (Table 3). ", "section_name": "Discussion", "section_num": "4." }, { "section_content": "Cancer cachexia involves catabolic, mostly inflammatory mediators, that not only trigger skeletal muscle wasting but may also promote cardiac cachexia.This includes myocardial atrophy, dysfunction, and related remodeling that critically compromises cancer patients' prognosis and quality of life [17][18][19].The present study aimed to evaluate the role of MAO-A, a novel potential therapeutic target in CC, in the context of cardiac cachexia [38]. Using an established triple transgenic mouse model with orthotopic PDAC [41,42] compared to WT mice, we demonstrate for the first time that PDAC (-related CC) is associated with a significantly higher protein expression of MAO-A in the left myocardium.As a mitochondrial source of H 2 O 2 , MAO-A (like MAO-B) contributes to oxidative stress in the myocardium as well as in skeletal muscle [27][28][29][30]33,37,43]. High MAO-A expression or activity is clearly implicated in several cardiac conditions [27,29,31,32] that benefit from MAO-A inhibition [34][35][36].As yet, MAO-A has not been addressed in cardiac cachexia; however, we recently reported, in the presently studied PDAC-related mouse model of CC, that MAO-A protein expression was significantly increased in skeletal (gastrocnemius) muscle.Furthermore, MAO-A inhibition with HH significantly attenuated transcripts of pro-inflammatory IL-1β in the gastrocnemius muscle and of the pro-atrophic E3-ligases atrogin-1 and MuRF1 in the soleus muscle without changing MAO-A protein expression levels [38].Unfortunately, this potentially anti-cachectic effect of HH failed to translate into a reversal of muscle skeletal wasting, likely due to a compromised neuromuscular junction as an adverse effect of HH.Given the principle difference in cardiac innervation, we presently hypothesized that HH treatment could offer a therapeutic option by attenuating possible CC-related upregulation of E3 ligases and upstream cytokine triggers responsible for myofibrillar breakdown in myocardium as well as skeletal muscle [17,44]. As a main finding, we observed a PDAC-related highly significant increase in myocardial atrogin-1+ cell fraction (%) that was significantly, though incompletely, reversed by the reversible selective MAO-A inhibitor HH.Notably, these opposing effects of PDAC and HH treatment were also well reflected in terms of atrogin-1+ cell density (n/mm 2 ).However, this may be more biased through possible cardiomyocyte atrophy (not assessed) than with normalization to the total count of nuclei, which was found to be similar among the four groups.Notably, atrogin-1 (and MuRF1) transcript levels were largely unaffected by PDAC or HH treatment, potentially indicating feedback regulation at high atrogin-1 protein abundance.Nevertheless, these data suggest that enhanced H 2 O 2 production through increased MAO-A expression or (e.g., catecholamine-induced) activity may well contribute to upregulation of pro-atrophic atrogin-1 in cardiomyocytes of PDAC-bearing mice, since MAO inhibition mostly reversed atrogin-1+ expression, which was immunolocalized within cardiomyocytes.In support of such a potentially causal role for MAO-A in atrogin-1 upregulation, there was the finding of a significant and positive relationship between percentage atrogin-1+ cells (r = 0.687 p = 0.001) and MAO-A protein expression, irrespective of HH treatment.Notably, HH did not affect MAO-A protein or RNA expression but was presently assumed to reduce atrogin-1+ cells via inhibition of MAO-A activity (not measured). Atrogin-1 is a well-established pro-atrophic [16,17,44] or anti-hypertrophic [45,46] myocardial ubiquitin E3 ligase that is under converging control of the myostatin-ActRIIB-Smad 2/3-, IGF-1-Akt-, or NF-κB-dependent FoxO3 transcription factor [8,12,13,47,48], which clearly regulates the cardiomyocyte size [49].For example, increased RNA and protein expression of atrogin-1 together with MuRF-1 were found with myocardial or skeletal muscle wasting in a Lewis lung cancer mouse model and attributable to NF-κB activation due to increased Iκκβ and phospho-p65 expression [17], as NF-κB inhibition via luteolin largely reversed E3 ligases and losses in cardiac and skeletal muscle mass.In the present study, NF-κB signaling in response to H 2 O 2 may well convey the observed atrogin-1 upregulation [50,51] since it can be inhibited together with cytokine expression by HH [52], as presently supported by the reversal of upregulated IL-1β and TNF.These cytokines indeed trigger E3 ligase upregulation and, in turn, reinforce their transcription via NF-κB activation [5,8,12,15,51,[53][54][55][56].Systemic or local upregulation of TNF may have triggered oxidative stress and further cytokine release, all of which can be attenuated by antioxidants [57] and likely by inhibition of MAO-A activity, which notably itself responds to oxidative stress [44].In addition to NF-κB, the complex downstream effects of PDAC may also involve inhibition of the IRS-AK pathway by TNF or Smad 2, 3 or other factors [8,12,58].Notably HH treatment also led to marked reversal of the upregulated SOCS3 transcripts to below WT (control) levels.SOCS3 expression can occur in response to cytokine-triggered STAT3 signaling or H 2 O 2 and likely contributes to proteasomal degradation [9, [59][60][61] and cardiac remodeling, e.g., after myocardial infarction [62].Although interconnected, all pro-cachectic pathways involved appear to finally converge in atrogin-1 expression, which presently was effectively reduced by HH treatment.Thereby, atrogin-1 may co-activate its own transcription factor via Akt, what can be considered to be a positive feedback mechanism [45].Notably, however, an overly simplistic total inhibition of H 2 O 2 may not be desirable with all signaling steps, as certain prolonged high levels of H 2 O 2 may promote cardiac hypertrophy rather than atrophy [63] and the anabolic IGF-1-Akt pathway may be activated by H 2 O 2 , at least in myotubes [64]. Regarding the observed (2.08-fold increase) increased fraction of COX2-expressing cells with PDAC, in accordance with transcript levels, one may assume that a PDAC-related infiltration (1.57-fold increase) of CD68+ cells (macrophages) into the myocardium occurs, which may locally trigger COX2 expression in various cells including cardiomyocytes.Why the HH-induced reduction of COX2-transcript levels does not translate into reduced COX2-protein expression remains unclear at present.Notably, COX2 upregulation may have a time-dependent cardioprotective role under conditions of H 2 O 2 treatment [65] and may be required for hypertrophy, at least in skeletal muscle [66]. To our knowledge, myocardial capillary rarefication has not been previously reported in the context of cardiac cachexia but may be highly relevant for cardiac dysfunction.The significant decrease in capillary density by -17% may even underestimate the actual loss in capillary contacts per fiber, as any cardiomyocyte atrophy will enhance, per se, capillary density.The factors behind PDAC-related capillary losses despite stable VEGFA expression presently remains elusive.However, they may include the observed reduction in KDR that, like capillary density, failed to improve with HH treatment.A high PDAC-related TNF expression may, in theory, convey an anti-angiogenic effect [67]; however, this should involve lower VEGFA expression levels, which were presently not observed.Moreover, the HH-related decreases in TNF were presently not associated with improved capillarization.However, as a note of caution, any anti-atrophy effect on fiber size via HH treatment would result in a decreased capillary density, i.e., mask any potential neo-angiogenesis.As COX2 plays a role in myocardial angiogenesis, its regulation with PDAC and HH (downregulated on the RNA level) may warrant further studies including the function of inflammatory macrophages [68,69]. In addition to MAO-A, HH treatment may also inhibit the x-chromosomal multifunctional dual-specificity tyrosine phosphorylation-regulated kinases 1A (DYRK1A) and 1B (DYRK1B /MIRK) [70].Like MAO-A, DYRK1A and 1B may be overexpressed in heart failure and impair hyperplasia and mitochondrial function, respectively [71,72].However, in contrast to MAO-A, DYRK1B may have an antioxidative role promoting myogenesis and autophagy, at least in skeletal muscle [73,74].Thus, HH inhibition of MAO-A and DYRK1B may result in differential and not thoroughly beneficial effects.This may also be true for DYRK1A, which promotes an angiogenic response by interfering with VEGFdependent activation of the Ca 2+ /nuclear factor of activated T-cells (NFATs) pathway in endothelial cells [75].DYRK1A inhibition may thus be anti-angiogenic and oppose any potential pro-angiogenic effect of MAO-A inhibition [40], in line with the failure of HH to improve capillarization.However, DYRK1A inhibition may facilitate cardiac repair and function by increasing cardiomyocyte cycling/hyperplasia following ischemia-reperfusion stress [71].Nevertheless, capillary rarefication may generally arise from cardiac detraining/inactivity as a result of both CC-related and the recently reported HH-related skeletal muscle atrophy [38]. As a limitation, the presently studied HH intervention in PDAC and WT mice was not sham-controlled by a daily i.p. application of the HH medium, which by itself might have had an effect.However, the selective reversal of PDAC-related increases in atrogin-1, TNF, or CD68 (as reflected by the significant interaction of PDAC and HH in ANOVA), supported the hypothesis that inhibition of the overexpressed MAO-A may be the main factor behind such beneficial changes.Together with further limitations like large variability in MAO-A expression, pooled PCR analyses, some gender imbalance between the groups, and the limited sample size associated with the risks of type 1 and type 2 errors, our present data may be considered hypothesis-generating.In view of lacking therapeutic options of CC, the present data call for larger controlled studies with consideration of gender, systemic inflammation, physical activity, cardiomyocyte size, myocardial muscle mass, and related functional readouts as well as of tumor mass and staging [9,76]. In summary, preliminary evidence is presently provided for MAO-A as a potential therapeutic target in cardiac cachexia, since inhibition of MAO-A as a source of H 2 O 2 by HH reverses high left myocardial expression of atrogin-1 and of the pro-cachectic cytokines IL-1β and TNF as well as SOCS3 and COX2 on the RNA level.The functional significance of such anti-atrophic/-inflammatory HH effects remained open at present.The novel finding of PDAC-related myocardial capillary rarefication, which did not improve with HH treatment, warrants timely clarification regarding its mechanism and functional relevance. ", "section_name": "Discussion", "section_num": "4." } ]	[ { "section_content": "We gratefully acknowledge the expert laboratory assistance of Claudia Keppler, Christa Merte-Grebe, Aninja Baier, Elke Völck-Badouin, Irmgard Dammshäuser.and Michael Dreher. ", "section_name": "Acknowledgments:", "section_num": null }, { "section_content": "The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. ", "section_name": "Data Availability Statement:", "section_num": null }, { "section_content": "Author Contributions: Conceptualization, W.H. and E.P.S.; methodology, K.S., A.A., G.A.B., H.S. and E.P.S.; formal analysis, A.A., K.S., G.A.B., H.S. and W.H.; writing-original draft preparation, W.H., K.S. and A.A.; writing-review and editing, K.S., R.K., E.P.S. and W.H.; supervision, E.P.S. and W.H.; project administration, V.F. and R.K.; funding acquisition V.F., W.H. and R.K.All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the University Clinics of Giessen and Marburg (UKGM) according to their cooperation agreement (Project/grant number 44/2011) and 'Open Access Publishing Fund' of the Philipps University, Marburg with support of the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation). The animal study protocol was approved by the Regional Commission Giessen (RP, Giessen, Hessen, Germany, MR 20/11-Nr.70/2009)and conducted according to German animal welfare law and the European legislation for protection of animals used for scientific purposes (2010/63/EU) as well as to regulations for animal experiments of the Philipps University of Marburg. Informed Consent Statement: Not applicable. The authors declare no conflicts of interest. ", "section_name": "Institutional Review Board Statement:", "section_num": null }, { "section_content": "Author Contributions: Conceptualization, W.H. and E.P.S.; methodology, K.S., A.A., G.A.B., H.S. and E.P.S.; formal analysis, A.A., K.S., G.A.B., H.S. and W.H.; writing-original draft preparation, W.H., K.S. and A.A.; writing-review and editing, K.S., R.K., E.P.S. and W.H.; supervision, E.P.S. and W.H.; project administration, V.F. and R.K.; funding acquisition V.F., W.H. and R.K.All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the University Clinics of Giessen and Marburg (UKGM) according to their cooperation agreement (Project/grant number 44/2011) and 'Open Access Publishing Fund' of the Philipps University, Marburg with support of the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation). ", "section_name": "", "section_num": "" }, { "section_content": "The animal study protocol was approved by the Regional Commission Giessen (RP, Giessen, Hessen, Germany, MR 20/11-Nr.70/2009)and conducted according to German animal welfare law and the European legislation for protection of animals used for scientific purposes (2010/63/EU) as well as to regulations for animal experiments of the Philipps University of Marburg. Informed Consent Statement: Not applicable. ", "section_name": "Institutional Review Board Statement:", "section_num": null }, { "section_content": "The authors declare no conflicts of interest. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.1371/journal.ppat.1002560	Redundant Notch1 and Notch2 Signaling Is Necessary for IFNγ Secretion by T Helper 1 Cells During Infection with Leishmania major	The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+) T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+) T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4(+) T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre)) were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre) mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+) T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+) T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.	[ { "section_content": "Following activation by pathogens, naı ¨ve CD4 + T cells can differentiate into several functionally distinct T helper (Th) subsets, defined by the cytokines they secrete.CD4 + Th1 cells secrete IFNc as a signature cytokine and the transcription factor T-bet is essential for their differentiation.Although cytokines such as IL-12 contribute to Th1 cell differentiation, Th1 cells can be generated in the absence of cytokine signaling, demonstrating a role for other molecules in this process.Among these are Notch receptors and their ligands (Reviewed in [1,2]).Notch signaling plays crucial roles in binary cell fate decisions in many developmental systems including the development and differentiation of immune cells.In mammals, there are four Notch receptors (Notch1-4) that are activated by five ligands (Jagged (Jag) 1, and 2, and Delta-like (Dll) 1, 3, and 4).Upon interaction with its ligand, the active intracellular domain of Notch (NICD) is released from the membrane by proteolytic cleavages and translocates into the nucleus.Once there, NICD can form a complex with recombination signal-binding protein-J (RBP-Jk), converting it to an activator of transcription (canonical Notch signaling).Alternative-ly, NICD could interact with members of the NF-kB pathway (non-canonical Notch signaling) [3].In the T cell lineage, the Notch1 receptor is essential for the development of ab T cells [4], and Notch plays a poorly understood role in the differentiation of peripheral Th cell subsets (reviewed in [1,5]). The importance of Notch signaling during CD4 + Th1 differentiation and its correlated resolution of pathogen infection is currently unclear.Inhibitors of c-secretase impairing Notch signaling prevented Th1 differentiation in vitro and in vivo, potentially through the blocking of T-bet expression [6].Blocking of the Notch3 receptor using antisense N3 DNA also blocked Th1 differentiation in vitro [7].In contrast, T cell-specific expression of dominant negative mastermind-like protein (MAML1), which is needed for RBP-Jk-dependent Notch signaling, or T cell specific ablation of Notch1 or RBP-Jk did not have an impact on Th1 differentiation in vitro [8] nor in vivo [9,10]. The role of Notch ligands on dendritic cells instructing Th1 differentiation is also debated.Dll1 and/or Dll4 expression is upregulated in vitro on APCs encountering pathogens driving a CD4 + Th1 response [8,11,12].Interaction of Notch with Dll1 promoted Th1 differentiation during Leishmania major infection [7]. Furthermore, Dll4 expression on DC was shown to induce Th1 cell differentiation in an IL-12-independent way [11].On the contrary, Dll1, Jag1 and Jag2 were shown to be insufficient to instruct the differentiation of Th1 or Th2 CD4 + cells in absence of polarizing cytokines in vitro, suggesting that the induction of selective ligands by pathogens may not exert a direct influence on T helper differentiation [13,14]. Altogether, these studies indicate a role of Notch in CD4 + Th1 differentiation, but it is not clear yet which member and how each member of this family contributes to this process during infection with pathogens.Most of the above studies investigated the role of Notch using total inhibition of Notch signaling, but the individual contribution and potential crosstalk of individual Notch receptors during infections with pathogens inducing CD4 + Th1 cells has not been investigated. Here, mice carrying a T cell specific deletion of Notch1 (N1 DCD4Cre ), Notch2 (N2 DCD4Cre ) or both Notch1 and Notch2 (N1N2 DCD4Cre ) on a resistant C57BL/6 genetic background were infected with L. major to study the importance of Notch receptors in Th1 differentiation and resolution of the infection.We show that Notch signaling through either N1 and/or N2 induces the secretion of IFNc by CD4 + Th1 cells.Moreover, using mice with T cell-specific ablation of RBP-Jk (RBP-J DCD4Cre ), we show that Th1 differentiation is induced mainly by non-canonical (RBP-Jkindependent) Notch signaling.Collectively, our data indicate that Notch signaling drives the differentiation of L. major-specific IFNcsecreting Th1 cells required to mount an efficient immune response against this parasite. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "To investigate Notch function in Th1 cell development we infected mice with L. major, a parasite promoting a predominant Th1 immune response in most strains of mice including C57BL/6 [15].Of the four Notch receptors, only N1 and N2 are expressed in activated T cells [8,16].Thus, to investigate the effect of T cell ablation of these two receptors (N1N2 DCD4Cre ) on CD4 + Th1 differentiation and the consequent resolution of L. major infection, N1N2 DCD4Cre and control N1N2 lox/lox mice on the L. major resistant C57BL/6 genetic background were inoculated with the parasite.In contrast to N1N2 lox/lox control mice that developed a small self-healing lesion, N1N2 DCD4Cre were unable to heal their lesions (Figure 1A).In addition, L. major-infected N1N2 DCD4Cre mice failed to control parasite load at the site of parasite inoculation (Figure 1B) and L. major disseminated to the lymph nodes and spleen (Figure 1C,D). The impact of the absence of Notch on T cells in the differentiation of CD4 + IFNc-secreting Th1 cells was assessed six weeks after infection.N1N2 DCD4Cre and control draining lymph node (dLN) cells were restimulated in vitro with UV-irradiated L. major and cytokine levels measured.Strikingly, secretion of IFNc was abrogated in supernatants of N1N2 DCD4Cre dLN cells, while high levels of this cytokine were measured in dLN from infected control mice (Figure 1E).Similarly low IL-4 levels were measured in each group (Figure 1F).IL-13 and IL-5 were found predominantly in N1N2 DCD4Cre dLN cells (Figure 1G).The persistence of parasites in the dLN of L. major infected N1N2 DCD4Cre but not N1N2 lox/lox mice was sufficient to induce IL-13 and IL-5 secretion by T cells (Figure 1G), albeit at a lower level than that reached following stimulation with exogenous addition of L. major.These results show that Notch signaling contributes to the generation of IFNc-secreting CD4 + T cells, which are essential in the control of parasite load and lesion size.The absence of Notch expression on T cells, while preventing IFNc secretion, favored the development of IL-13-and IL-5secreting cells. ", "section_name": "Notch affects the development of a protective L. majorspecific Th1 cell response", "section_num": null }, { "section_content": "N1 DCD4Cre mice are able to develop a protective Th1 response in response to L. major inoculation [9].The inability of L. majorinfected N1N2 DCD4Cre mice to develop a protective Th1 immune response suggested that N2 could be the receptor involved in Th1 differentiation.To investigate this, N2 DCD4Cre and N2 lox/lox control mice were infected with L. major and evolution of lesion size and development of immune response were compared to that developing in N1 DCD4Cre , and N1N2 DCD4Cre infected mice.N2 DCD4Cre mice were able to control their lesion size (Figure 2A) and parasitemia (Figure 2B) as well as N1 DCD4Cre and control mice, unlike N1N2 DCD4Cre mice.To analyze their immune response, cytokine secretion by dLN cells was analyzed.L. majorinfected N2 DCD4Cre dLN cells secreted similar levels of IFNc than L. major-infected N1 DCD4Cre and control mice.Low levels of IL-4, IL-5 and IL-13 were similarly measured in their dLN cells (Figure 2C).N1N2 DCD4Cre mice are susceptible to L. major infection and fail to induce IFNc secretion by CD4 + T cells, indicating that expression of either N1 or N2 is sufficient to induce CD4 + Th1 differentiation in N2 DCD4Cre or N1 DCD4cre mice, respectively.To investigate if following parasite inoculation, compensatory expression of one or all of the Notch receptors on T cells could occur, dLN cells of L. major infected N1 DCD4Cre or N2 DCD4Cre mice were stimulated in vitro with L. major, and Notch expression on their CD4 + T cells measured by FACS.N1 expression was significantly and similarly expressed in both N2 DCD4Cre and N2 lox/lox CD4 + T cells (Figure 3A).Low levels of N2 surface expression were induced following restimulation of control dLN T cells with L. major, however, a significantly higher induction of N2 was measured in L. major-activated N1 DCD4cre CD4 + T cells (Figure 3B), suggesting that compensatory mechanisms allow increased N2 expression in ", "section_name": "N1 or N2 expression on T cells is sufficient to drive Th1 cell differentiation", "section_num": null }, { "section_content": "Infection with protozoan parasites of Leishmania species results in a spectrum of local or systemic diseases in humans and mammals.Overall, leishmaniasis afflicts around 12 million individuals in 88 countries worldwide.Cutaneous leishmaniasis is the most prevalent form of the disease.In order to better understand the complex molecular pathways leading to protection against the cutaneous form of the disease, we used the Leishmania major mouse model.Most mouse strains control L. major infection due to the development of a Th1 response, leading to secretion of IFNc by T cells which promotes healing and resistance to reinfection.Notch signaling is a very conserved pathway in the regulation of cell differentiation and cell fate decision.However the contribution of Notch receptors in the response to parasite infection is not clear.In this study, we infected mice that do not express Notch1 and Notch2 receptors on the surface of their T cells.We show that these Notch receptors are key players in the development of a protective Th1 immune response against L. major.These results contribute to the understanding of the mechanisms involved in the development of a protective response against pathogens.absence of N1.No expression of N3 and N4 mRNA or proteins was detectable on T cells of all genotypes, in contrast to positive control cells (Figure 3C and data not shown).Altogether, these data reveal that signaling through either N1 or N2 is sufficient for the generation of functional Th1 cells following infection with L. major, and that in absence of N1 compensatory higher expression of N2 is measured on T cells. ", "section_name": "Author Summary", "section_num": null }, { "section_content": "Draining LN CD4 + T cells of L. major-infected N1N2 DCD4Cre mice fail to secrete IFNc but release high levels of IL-13.This cytokine has been associated with susceptibility to L. major infection, mostly by preventing the classical activation of macrophages by IFNc [17].To investigate if susceptibility of N1N2 DCD4Cre mice resulted from a lack of IFNc secretion and/or from the presence of high levels of IL-13, IL-13 was neutralized with an anti-IL-13 mAb after inoculation of L. major in N1N2 DCD4Cre and N1N2 lox/lox control mice.No effect was observed in lesion development and parasite control in IL-13depleted N1N2 DCD4Cre mice, that developed unhealing lesions similar to mice treated with PBS (Figure 4A).Similar low levels of IFNc were measured in isolated CD4 + T cells of mice depleted or not of IL-13 (Figure 4B).The efficacy of the anti-IL-13 treatment was confirmed by measuring dLN levels of Fizz1 and Ym1 expression, two markers of alternative macrophage activation.The mRNA levels of both markers were decreased in dLN of anti-IL-13-treated N1N2 DCD4Cre mice (Figure 4C).Collectively, these data demonstrate that the non-healing phenotype measured in N1N2 DCD4Cre mice results primarily from the decreased IFNc secretion by CD4 + T cells which does not allow activation of macrophage to kill the intracellular parasites.The high levels of IL-13 which induce alternative macrophage activation do not play a critical role in the failure of macrophages to kill the parasites, as in absence of IFNc, macrophages are already not classically activated. ", "section_name": "Susceptibility to L. major infection in absence of Notch on T cells results mainly from lack of IFNc secretion", "section_num": null }, { "section_content": "We then investigated if the impaired IFNc secretion measured in N1N2 DCD4Cre CD4 + T cells could result from defective in vitro proliferation.To this end, dLN cells of L. major-infected N1N2 DCD4Cre and control mice were stained with CFSE and restimulated for 72 h with L. major.CD4 + T cells of both N1N2 DCD4Cre and control proliferated in response to the parasite.N1N2 DCD4Cre showed a slightly lower CD4 + T cell proliferation compared to that of N1N2 lox/lox CD4 + T cells but the difference was not statistically significant (Figure 5A).Despite comparable proliferation, IFNc was not secreted in response to L. major stimulation.However, high levels of intracellular IFNc were measured by FACS in N1N2 DCD4Cre CD4 + T cells restimulated with L. major for 72 h, in absence of PMA-ionomycin stimulation (Figure 5B).To further determine at which level the absence of N1 and N2 on T cells affects secretion of IFNc in the dLN of L. majorinfected mice, mRNA levels of IFNc and T-bet, the major transcription factor of Th1 cells, were measured ex vivo on FACS sorted CD4 + T cells 3 weeks after infection.Reduced levels of secreted IFNc did not result from impaired transcription of T-bet or IFNc mRNA as demonstrated by higher levels of both T-bet and IFNc mRNA measured in N1N2 DCD4Cre CD4 + T cells compared to those measured in CD4 + T cells of control mice (Figure 5C).No defect in activation status or in CD4 + T cell number was measured in dLN cell N1N2 DCD4Cre mice (Figure S1).IFNc signaling is mediated by STAT1 phosphorylation and IFNc was reported to signal to the majority of cells throughout the dLN during a Th1 response after T. gondii infection [18].To further show that secretion of IFNc is impaired in CD4 + T cells during infection, we measured STAT1 phosphorylation in dLN CD4 + T cells of L. major-infected N1N2 DCD4Cre mice.STAT1 phosphorylation was detected in CD4 + T cells of control mice while only minimal STAT1 phosphorylation was measured in CD4 + T cells of N1N2 DCD4Cre dLN cells (Figure 5D).These data confirm that in absence of N1 and N2 on T cells, IFNc secretion by CD4 + T cells is impaired, thereby preventing IFNc-induced STAT1 phosphorylation occurring in vivo.The impairment of IFNc secretion is antigen-specific and not due to an intrinsic secretion default in N1N2 DCD4Cre mice as revealed by the high levels of IFNc detected by intracellular staining following TCR-independent T cell stimulation (PMA-ionomycin) ex vivo (Figure 5E).In addition, defective secretion could be overcome in vitro by antibodymediated CD3 crosslinking stimulation (Figure S2A). In the same line, naı ¨ve N1N2 DCD4Cre CD4 + T cells stimulated in vitro in the presence of standard Th1 polarizing conditions followed by stimulation with anti-CD3/CD28 for 48 hours were able to develop into IFNc-secreting cells (Figure S2B).In contrast, the strong increase in IL-13 and IL-5 mRNA levels measured in dLN of L. major-infected mice (Figure 5F) correlated with the high secretion levels of these cytokines in L. major-stimulated CD4 + T cells.Altogether, these data reveal that following inoculation of L. major, absence of N1 and N2 on T cells prevents the release of IFNc by CD4 + T cells, favoring the differentiation of IL-13-and IL-5-secreting cells.This effect is obscured in vitro by exogenous addition of high amounts of cytokines and/or antigen-non specific activation of T cells. ", "section_name": "Notch signaling prevents the release but not the transcription of IFNc by CD4 + dLN cells", "section_num": null }, { "section_content": "Mice with dominant negative MAML (DNMAML) protein preventing the canonical transcriptional activation by all four Notch receptors were previously reported to be able to control infection with L. major and to have normal levels of IFNc in their dLN CD4 + T cells [10].Different strains of L. major may induce distinct type of T helper immune response [19].To further insure that the different outcomes on the differentiation of Th1 cells measured in theirs (L.major Friedlin) and the present studies (L.major LV39) were not due to differences in the L. major strains used, N1N2 DCD4Cre mice were infected with two other L. major strains (Friedlin or IR175).N1N2 DCD4Cre mice infected with these two L. major strains failed to develop an efficient Th1 response with decreased secretion of IFNc and increased secretion of IL-13 and IL-5 by their dLN T cells and high intralesional parasite load (Figure S3).These data show that N1 and N2 are required for Th1 differentiation following infection with different strains of L. major. Having ruled out a potential effect due to the strain of Leishmania used, the lack of effect of DNMAML on Th1 differentiation [10] suggested that Notch signaling may not drive Th1 cell differentiation through the NICD-MAML-RBP-Jk transcriptional activation complex.To investigate if the requirement of Notch signaling for CD4 + Th1 differentiation and the associated resolution of the lesion could be RBP-Jk-independent, we infected RBP-Jk DCD4Cre and RBP-Jk lox/lox control mice with L. major.No difference in lesion development (Figure 6A) nor parasite control was measured between RBP-Jk DCD4Cre and control mice (Figure 6B).Furthermore, the development of CD4 + IFNcsecreting Th1 cells was normal, as revealed by high levels of IFNc secretion by dLN T cells, and low levels of IL-13 and IL-5 (Figure 6C).These results demonstrate that RBP-Jk-independent Notch signaling is required for CD4 + Th1 differentiation following L. major infection. ", "section_name": "Notch signaling driving CD4 + Th1 differentiation occurs in absence of the RBP-Jk transcription factor", "section_num": null }, { "section_content": "The development of Th1 cells, through their secretion of IFNc, contributes to a number of protective effects against many pathogens.Despite a growing understanding on the mechanisms leading to T helper differentiation these last years (reviewed in [20]), there are still unresolved issues including the identification of which receptor triggering leads to Th1 differentiation.The role of Notch in Th1 differentiation has been controversial, depending on the mode of activation/deactivation of Notch [1,5].Our data showing the crucial role of RBP-jk-independent Notch signaling in the differentiation of IFNc-secreting T cells help to reconcile discrepant results obtained using different loss or gain of function approaches that attributed or not a role for Notch signaling in Th1 differentiation. Here, using mice with ablation of Notch in their T cells, we demonstrate that expression of either N1 or N2 on T cells is necessary and sufficient for the differentiation of IFNc-secreting Th1 cells and the consequent control of infection.N1N2 DCD4Cre mice infected with L. major failed to mount a protective Th1 immune response while mice with single deletion of N1 (this study and [9]) or N2 in their T cells developed a protective Th1 immune response.In control mice, N1 is the only receptor expressed at significant levels at the surface of anti-CD3 and L. major-activated CD4 + T cells.There appears to be functional redundancy of N1 and N2 in driving CD4 + T helper 1 differentiation.Expression of N2 is low in activated T cells of L. major-infected control mice, but compensatory increased N2 expression was measured in absence of N1 expression.This suggests that N1 is the main receptor involved in signaling leading to the induction of IFNc secretion by T cells following infection with L. major, but that in absence of N1, higher levels of N2 can compensate its absence.Functional redundancy of N1 and N2 was previously suggested in N1IAS mice that had decreased but not abrogated IFNc secretion [6], however the expression of Notch receptors was not assessed in that study.Of note, we show here that N2 is the only receptor that could functionally substitute for N1 in driving Th1 differentiation in vivo, and T cell expression of N3 or N4 were not detected in presence or absence of N1 and/or N2 in CD4 + T cells of L. majorinfected mice.In addition, N1N2 DCD4Cre mice do not control infection revealing that N3 and N4 are not functionally redundant in driving IFNc secretion by CD4 + T cells.Overexpression of N3 intracellular domain (N3IC) in T cells was previously reported to induce IFNc secretion in vitro following anti-CD3 activation, while overexpression of N1IC did not, suggesting that N3 could be involved in Th1 differentiation [7].Together with our reported increased expression of N2 in absence of N1, these results show that individual Notch receptors may potentially drive IFNc secretion by CD4 + T cells, but during L. major infection N1, and to a lesser extent N2 appear to be the only receptors involved in driving Th1 differentiation. It was reported that N1 could regulate Th1 cell differentiation by interacting with CSL sequences present in the promoter of the Tbx21 gene which codes for T-bet, the master regulator of Th1 cell differentiation [6].However, in another study, Notch was not found to reside at the Tbx21 promoter [21].In addition, we show here that mice with specific ablation of RBP-Jk in their T cells, unlike N1N2 DCD4Cre mice, are able to mount a Th1 response and heal their lesion following infection with L. major.These results show that the Notch signaling playing a major role in the differentiation IFNc-secreting cells following infection with L. major occurs in absence of a CSL/RBP-Jk-transcription complex.In line with these data, it was previously reported that mice that conditionally expressed a dominant negative MAML protein (DNMAML) and thereby are deprived of RBP-Jk-mediated transcriptional activation of all Notch receptors, were able to develop a protective Th1 immune response following L. major infection [10].The present results show that Notch receptors are crucial to trigger secretion of IFNc by CD4 + T cells in a CSL/ RBP-Jk-independent manner. The nature of a CSL/RBP-Jk-independent Notch pathway is complex and not yet defined [22].It has been reported that Notch can associate with the nuclear factor kB (NF-kB) proteins p50 and p65.Furthermore, Notch1-NF-kB complexes could be immunoprecipitated from the Ifng promoter despite the lack of consensus binding sites for RBP-Jk in the promoter of this gene [23].This suggested that N1ICD could contribute to Th1 differentiation in a RBP-Jk-independent way leading to the hypothesis of a connection between Notch, NF-kB and Th1 differentiation [1,5].Of note, NF-kB p50 is required for optimal Th1 development and L. major-infected NF-kB1 knockout mice show a susceptible phenotype associated with defective secretion of IFNc [24].However in that study, failure to secrete IFNc was linked to a major defect in CD4 + T cell proliferation measured in vitro, while we did not detect such impairment of CD4 + T cell proliferation in Notch deficient CD4 + T cells.Thus Notch may interact with distinct transcription factors involved in the secretion of IFNc by Th1 cells and further molecular studies will be needed to determine the nature of these factors as well as the molecular mechanisms involved in the RBP-Jk-independent Notch signaling during Th1 differentiation. Notch signaling is required for proper secretion of IFNc by CD4 + Th1 cells following antigen-specific stimulation.Interestingly, increased expression of T-bet and IFNc mRNA was measured in dLN CD4 + T cells of L. major-infected N1N2 DCD4Cre mice revealing that Notch signaling does not prevent the differentiation of ''competent'' CD4 + Th1 cells [25], but appears to act downstream of it.The increase in T-bet and IFNc mRNA measured in CD4 + N1N2 DCD4Cre T cells suggests that intact Notch signaling regulates the extent transcription for these genes in vivo.Low levels of STAT1 phosphorylation in dLN CD4 + T cells confirmed that only very small amounts of IFNc protein, maybe released by NK cells, are present in the dLN of L. major-infected N1N2 DCD4Cre mice.In absence of IFNc, mice on the resistant C57BL/6 genetic background develop a Th2 immune response, with high levels of IL-4, IL-5 and IL-13 cytokines [26].Accordingly, impaired secretion of IFNc by CD4 + T cells of L. major-infected N1N2 DCD4Cre mice allowed the differentiation of IL-5-and IL-13-secreting Th2 cells.However, no increased secretion of IL-4 was measured in CD4 + T cells of N1N2 DCD4Cre L. majorinfected mice, in line with the previously reported crucial importance of Notch in driving IL-4 secretion by CD4 + T cells [8,21,27].Interestingly, absence of Notch did not impair the differentiation of other Th2 effector T cells, suggesting that following L. major infection, Notch is acting directly on the IL-4 promoter, as previously reported [8], and does not affect the differentiation of IL-13-and IL-5-Th2 secreting cells. Notch signaling is resulting from an interaction between Notch receptors and ligands on antigen presenting cells.Several ligands have been linked to Th1 differentiation in distinct experimental models of disease and Delta-like ligands have been linked to Th1 differentiation or impaired Th2 differentiation [8,11,12,28,29].Dll1 stimulation was shown to trigger Th1 development following L. major infection, but it was not determined which Notch receptor was interacting with this ligand [7].The present study shows that either N1 or N2 could be interacting with Dll1.Whether other Notch ligands are involved in Notch signaling during Leishmania infection remains to be investigated.Interestingly, it was reported recently that within the 6q27 gene cluster, the Dll1 gene was linked to susceptibility to visceral leishmaniasis, and reduced Dll1 expression was measured in VL patients in Sudan, Brazil, and Northen India [30].Thus genetic regulation of one of the Notch ligand, such as the downregulation of Dll1 expression appears to have major consequences on susceptibility to VL. Together with the present study, it reveals that a proper regulation of the Notch signaling pathway during infection with Leishmania parasites is essential for the development of a protective response against these parasites. Further understanding of the mechanisms by which Notch receptors regulate the differentiation of IFNc-secreting Th1 cells as well as the ligands involved in this process should contribute to the development of new vaccines and immunotherapeutic targets towards Leishmania pathology, as well as in other infections requiring protective IFNc-secreting CD4 + Th1 immune response. ", "section_name": "Discussion", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "This study was carried out in strict accordance with the recommendations in the Guide for the care and use of laboratory animals from the Department of security and Environment (DSE) from the state of Vaud, Switzerland.The protocol has been approved by the Ethics and Veterinary office regulations of the state of Vaud (SAV), Switzerland.Our laboratory has the administrative authorization numbers 1266-3, -4 and -5. ", "section_name": "Ethics statement", "section_num": null }, { "section_content": "The following T cell specific gene-targeted mice were generated by crossing floxed Notch1 [31], floxed Notch2 [32], double floxed Notch1-Notch2 or floxed RBP-Jk [33] mice, with mice carrying the CD4Cre transgene [34].N1 lox/lox , N2 lox/lox , N1N2 lox/lox and RBP-Jk lox/lox littermates were used as controls.All mice were on a C57BL/6 genetic background.T cell-specific deletion of Notch and RBP-Jk was verified for each strain by PCR.All mice were bred and maintained under pathogen-free conditions in the animal facility at the CIIL, Epalinges, Switzerland. ", "section_name": "Mice", "section_num": null }, { "section_content": "Leishmania major LV39 (MRHO/Sv/59/P strain) was used.Mice were infected s.c. with 3610 6 stationary phase L. major promastigotes in the footpad.Parasite load was assessed by limiting dilution analysis (LDA).Treatment with CNTO 134, a rat antimouse IL-13 mAb [35], a gift from Centocor, Inc, was initiated either six days or 21 days after infection, with injection of 500 mg i.p., once weekly in N1N2 DCD4Cre mice, while a control group similarly infected was treated with control IgG or PBS.As no biological differences were observed between L. major-infected mice treated with control IgG or PBS, PBS was used as control vehicle for CNTO 134. ", "section_name": "Parasites and infections", "section_num": null }, { "section_content": "Draining lymph node cells were cultured 6 UV-irradiated L. major promastigotes or anti-CD3 (clone 145-2C11, eBioscience) for 72 h.CD4 + T cells were isolated by MACS (Miltenyi Biotec), and cultured in the presence of irradiated C57BL/6 splenocytes.For in vitro experiment, naı ¨ve CD4 + CD62L + T cells were isolated by MACS and cultured as previously described [9].The cytokine content of the cell supernatant was measured by ELISAs.IFNc with a limit of detection of 10 IU/ml.IL-4, IL-5 (OptEIA from BD Biosciences) and IL-13 (DuoSet from R&D Systems) cytokines were analyzed with commercial kits.mRNA extraction and Real-Time PCR Extraction of total RNA was performed as previously described [36].Quantitative Real-Time PCRs were done using SYBR Green and a LightCycler system (Roche).Each cytokine mRNA was normalized to the relative hypoxanthine phosphoribosyltransferase (HPRT) endogenous mRNA expression, and represented as arbitrary units as described previously [36].Primers used were previously described [36,37,38]. ", "section_name": "Lymphocyte culture and cytokine assays", "section_num": null }, { "section_content": "Draining lymph node cells were isolated 3 weeks after L. major infection.Phosphorylation of STAT1 at tyrosine 701 (pY701) was detected by intracellular staining using an Alexa Fluor 488 conjugated anti-Stat1, PhosFlow Fix Buffer I and Perm Buffer III (BD Biosciences) according to manufacturer's instructions.CD4-PE-Cy5 and CD44-APC (eBiosciences) were used to stain cell surface.To assess Notch receptor expression, dLN cells were isolated and restimulated with UV-irradiated L. major for 16 hours.Cells were stained with anti-N1, anti-N2 biotinylated mAbs [16], followed by Streptavidin-PE, -APC (eBiosciences), PE-conjugated anti-N3 and anti-N4 (Biolegends).CD4 2 CD8 2 CD25 + thymocytes were used as positive control for N3 staining, and splenic CD8a + CD11c + dendritic cells were used as positive control for N4 staining.CD4 + T cells were gated using TCRb-APC and CD4-FITC (eBiosciences) mAbs.Dead cells were excluded using 7AAD (BD Pharmingen).For T cell proliferation, dLN cells were isolated 6 weeks post L. major infection and stained with CFSE (Molecular Probes).Cells were then restimulated 6 UV-irradiated L. major promastigotes for 72 h and analyzed by FACS.The following monoclonal Ab conjugates were used: CD4-PE-Cy5, CD8-APC, B220-Pe-TexasRed (eBioscience) and dead cells were excluded with DAPI.Intracellular IFNc was analyzed in dLN cells isolated 6 weeks post infection and restimulated with PMA (50 ng/ ml), ionomycin (500 ng/ml) and BrefelinA (1 mg/ml) for 4 h.Cells were stained for surface marker with the following mAb conjugates: CD4-PE-Cy5, CD8-APC (eBiosciences).Intracellular IFNc-PE was detected with an anti-IFNc-PE (BD Pharmingen).All analyses were performed on a FACS Calibur or a LSR II (Becton Dickinson) flow cytometers and data processed with FlowJo (TreeStar). ", "section_name": "Flow cytometry", "section_num": null }, { "section_content": "Data were analyzed using the Student's t-test for unpaired data. ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "", "section_name": "Supporting Information", "section_num": null } ]	[ { "section_content": "We thank, Dr Markus Mohrs for advice with the STAT1 assay and discussions, Dr P. Branigan (Centocor) for the anti-IL-13 mAb and discussions, Gre ´goire Simon for analysis of alternate macrophage activation, Dr Emma Fiorini and Anne Wilson for discussion, Dr Esther von Stebut for the L. major (Friedlin), Yazmin Hauyon-La Torre, and Tatiana Proust for technical expertise, Danny Labes for Cell sorting. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "This work was supported by grants to FTC from the Swiss National Science Foundation 3100030_129852, and 129700/1.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "Conceived and designed the experiments: FTC.Performed the experiments: FA SS MC FD EM.Analyzed the data: FA FTC.Contributed reagents/materials/analysis tools: UK FR HRM.Wrote the paper: FTC.Contributed to the design of the experiments: FA.Contributed to the editing of the manuscript: FA HRM FR. ", "section_name": "Author Contributions", "section_num": null } ]
10.1038/s41408-021-00528-x	Antibody responses after first and second Covid-19 vaccination in patients with chronic lymphocytic leukaemia	<jats:title>Abstract</jats:title><jats:p>B-cell chronic lymphocytic leukaemia (CLL) is associated with immunosuppression and patients are at increased clinical risk following SARS-CoV-2 infection. Covid-19 vaccines offer the potential for protection against severe infection but relatively little is known regarding the profile of the antibody response following first or second vaccination. We studied spike-specific antibody responses following first and/or second Covid-19 vaccination in 299 patients with CLL compared with healthy donors. 286 patients underwent extended interval (10–12 week) vaccination. 154 patients received the BNT162b2 mRNA vaccine and 145 patients received ChAdOx1. Blood samples were taken either by venepuncture or as dried blood spots on filter paper. Spike-specific antibody responses were detectable in 34% of patients with CLL after one vaccine (<jats:italic>n</jats:italic> = 267) compared to 94% in healthy donors with antibody titres 104-fold lower in the patient group. Antibody responses increased to 75% after second vaccine (<jats:italic>n</jats:italic> = 55), compared to 100% in healthy donors, although titres remained lower. Multivariate analysis showed that current treatment with BTK inhibitors or IgA deficiency were independently associated with failure to generate an antibody response after the second vaccine. This work supports the need for optimisation of vaccination strategy in patients with CLL including the potential utility of booster vaccines.</jats:p>	[ { "section_content": "Chronic lymphocytic leukaemia (CLL) is associated with profound immune dysregulation that progresses over the disease course.The underlying aetiology is multifactorial, with hypogammaglobulinaemia, impaired cellular immunity and therapy-related immunosuppression commonly observed [1].These perturbations in immunity predispose patients to an increased risk of infection and infection-related mortality remains a common cause of death [2].Vaccination against common infectious agents is of paramount importance in supportive care but vaccine-induced immune responses and associated clinical efficacy are often reduced in this patient group.SARS-CoV-2 is a novel coronavirus and has led to a global pandemic with over 3.2 million deaths to date.Several studies have shown increased rates of morbidity and mortality after SARS-CoV-2 infection in patients with CLL and this is exacerbated by the age of many patients with this condition [3][4][5]. Novel vaccines against Covid-19 have shown remarkable efficacy and are likely to play a major role in control of the current pandemic [6].BNT162b2 and ChAdOx1 utilise nucleosidemodified RNA or adenovirus-based platforms, respectively, and incorporate the SARS-CoV-2 spike protein as vaccine immunogen.Both vaccines are given as two doses with the BNT162b2 vaccine approved for a 3-week interval whilst clinical responses after ChAdOx1 are improved with a longer period between prime and boost [7].However, several countries, including the UK, have elected to adopt an 'extended interval' vaccination regimen of between 10-12 weeks between BNT162b2 doses in order to maximise population coverage after a single vaccine. Covid-19 vaccines offer the potential to provide patients with CLL with substantial clinical protection from SARS-CoV-2 infection but there is concern regarding the efficacy of vaccine responses in this group.Studies over many years have shown immune responses to vaccination are impaired in patients with B-CLL [8][9][10].This is seen most particularly in the response to novel immunogens [8,9].Attenuated vaccine-induced immunity is seen across many stages of the disease course but immune function deteriorates most particularly in heavily treated patients.Vaccine responses are also suppressed in patients who are undergoing treatment with BTK inhibitors [8,11].There is relatively little information to date regarding the efficacy of Covid-19 vaccination in patients with CLL.Examination of 44 patients following 2 doses of mRNA vaccines recently reported a response rate of 52% [12].Similarly, in a larger cohort of patients who had received the second BNT162b2 mRNA vaccine following a standard 3-week interval between doses, antibody responses were detected in 40%, with higher rates in patients with clinical remission after treatment, compared with a response rate of only 16% for patients on current treatment [13].No current data exists studying ChAdOx1 in patients with CLL or for those on extended interval schedules. Here we present an interim assessment of spike-specific antibody response in patients with CLL following BNT162b2 or ChAdOx1 vaccination.Due to usage of an 'extended interval' vaccine protocol within the UK for most patients to date, antibody responses have been assessed after single vaccination, with a smaller proportion assessed following the second dose.We show that immune responses are impaired in most patients and that IgA deficiency and current therapy with BTKi are independent risk factors for poor response. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "Patients with a diagnosis of CLL or small lymphocytic leukaemia (SLL) were recruited to the study.The work was performed under the CIA UPH IRAS approval (REC 20\\NW\\0240) and conducted according to the Declaration of Helsinki and good clinical practice.Informed consent was obtained in person or by remote consultation.Dates and subtype of SARS-CoV-2 vaccination were obtained together with self-reported information on disease stage and date of CLL diagnosis, CLL treatment and infection history.Infection history was considered 'positive' in cases of 2 or more serious or 3 or more respiratory infections in a 1-year period.Participants were asked about personal shielding and previous symptoms compatible with SARS-CoV-2 infection.Participant demographics can be found in Table 1. Samples were obtained following first or second vaccination.Local participants undertook a phlebotomy sample whilst those more distant donated a dried blood spot sample (DBS) from capillary blood after a finger prick (Fig. 1).A total of 93 healthy donor controls were recruited from local primary care networks. ", "section_name": "METHODS", "section_num": null }, { "section_content": "Serum was stored at -20 °C and defrosted prior to antibody analysis.IgG/ A/M antibodies specific to SARS-CoV-2 were detected using electrochemiluminescence assays on the automated Roche cobas e801 analysers based at Public Health England (PHE) Porton.Calibration and quality control were performed as recommended by the manufacturer.Anti-nucleocapsid protein (NP) antibodies were detected using the qualitative Roche Elecsys ® AntiSARS-CoV-2 ECLIA (COV2, Product code: 09203079190), whilst antispike (S) antibodies were detected using the quantitative Roche Elecsys ® Anti-SARS-CoV-2 S ECLIA (COV2 S, Product code 09289275190).Antinucleocapsid results are expressed as a cut-off index (COI) value, with a COI value of ≥1.0 considered positive for anti-nucleocapsid antibodies.Antispike results are expressed as units per ml (U/ml), with samples with a result of ≥0.8 U/ml considered positive for anti-spike antibodies within the fully quantitative range of the assay: 0.4-2500 U/ml.Samples >2500 U/ml were diluted further (1:10, 1:100, and 1:1000) to within the quantitative range. ", "section_name": "Roche Elecsys ® electrochemiluminescence immunoassay (ECLIA)", "section_num": null }, { "section_content": "Dried blood spot analysis was carried out by Clinical Immunology Service (University of Birmingham).Capillary blood samples were collected on DBS cards (Ahlstrom Munksjo) from participants remotely and stored at room temperature.Samples were eluted in 250 microlitres of 0.05% phosphatebuffered saline-Tween 20 (PBS, Oxoid Tween-20, Sigma-Aldrich) per blood spot and incubated overnight (12-16 h) before centrifugation (10,600 x g for 10 min).The DBS eluate was then applied to a pre-coated 96-well ELISA plate (The Binding Site (TBS), Birmingham, UK) containing stabilised trimeric SARS-CoV-2 spike glycoprotein and detecting IgG, IgA and IgM antibody isotypes [14].The performance characteristics for this assay were assessed in 162 non-hospitalised mild to moderate disease PCR-positive individuals and 707 presumed COVID-19 negative samples from pre-2019.Sensitivity was 96.3% (92.1-98.6)and specificity 99.3% (98.4-99.8).The ELISA output result was reported as a ratio relative to a monoclonal spikespecific calibration antibody standard and multiplied by the previously determined cut-off co-efficient to maintain batch-to-batch consistency, defined as 1.31.A positive result was classed as a ratio of 1 or more. ", "section_name": "Dried blood spot ELISA analysis", "section_num": null }, { "section_content": "Quantification of IgG, IgA and IgM was evaluated using the COBAS 6000 (Roche) at the University of Birmingham Clinical Immunology Service.For analysis of DBS eluates, levels were multiplied by 10.7 to account for dilution factor and following validation of paired DBS and healthy serum samples. ", "section_name": "Serum immunoglobulin measurement", "section_num": null }, { "section_content": "Data were tested for normality using Kolmogorov-Smirnov analysis.For comparative analysis of antibody titres between healthy donors and patients with CLL, Mann-Whitney U-tests were performed.Spearman rank correlation was used for comparing assay platforms and for correlating total serum Ig and time since diagnosis against anti-S responses.For comparison of anti-S response by CLL treatment groups, Kruskal-Wallis was performed with post-hoc Dunn's analysis and paired 1 st and 2 nd vaccine responses, Wilcoxon's matched-pairs signed rank test was used.Binary logistic regression of clinical variables was performed to test for associations with positive antibody response after the second vaccine.All analysis was performed using Graphpad prism v9.1.0for Mac (San Diego, California USA) aside from logistic regression for which SPSS Statistics v27.0 for Windows (Armonk, NY: IBM Corp.). ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "A total of 299 patients were enrolled in the study together with 93 age-matched healthy donors.The median age of the patient group was 69 years (IQR 63-74) and 159 of the CLL patients (53%) were male.A total of 154 patients had received the Pfizer-BioNTech mRNA vaccine (BNT162b2) whilst 145 received the AstraZeneca/Oxford ChAdOx1 adenovirus vaccine.286 patients (96%) were vaccinated with an 'extended interval' regimen of 10-12 weeks between the first and second vaccine.In this group blood samples were taken after the first vaccine in most cases in order to assess the immune response to single vaccine delivery.Thirteen patients (4%) were vaccinated according to the 'standard interval' for the mRNA vaccine of a 3-week time period between first and second dose.In this group samples were taken only after the second vaccine.Matched samples after first and second vaccine were available in 27 cases.The median time to sample collection following the first vaccination was 43 days (IQR: 36-52 days; n = 267) whilst the median time to sample collection following second vaccination was 18 days (IQR: 14-28; n = 55) (Fig. 1). One hundred and eighty one patients (61%) were at stage A and were untreated with 'watch and wait' monitoring.One hundred and eighteen (39%) had received treatment for CLL, of which 66 (22%) were actively being treated.The type and number of lines of therapy is shown in Table 1.Seventy-six patients (25%) reported a clinical history of frequent infections whilst 60 (20%) also reported previous hospital admission for infection.Fifty-five patients were on prophylactic antibiotics (18%) and 17 were on immunoglobulin replacement therapy (6%).The median time from diagnosis to sample collection was 79 months (IQR 39-142 months). Antibody responses following first vaccination are markedly reduced in patients with CLL Antibody responses in patients undergoing extended-interval vaccination were determined at 5-6 weeks after the initial vaccine.Analysis was performed on venepuncture serum samples (S1) in 86 patients and a simultaneous dried blood spot sample (DBS1) was also taken at the same time in 82 subjects.An additional 185 patients provided a DBS1 sample following the first vaccine such that a total of 267 samples were available for analysis. Twenty-nine of the 86 serum S1 samples (34%) gave a positive anti-spike antibody response using the Roche platform.This overall response rate was 2.8-fold lower than that of age-matched healthy donors where 94% were found to have detectable antibodies.Median antibody titres were also markedly reduced and were 104-fold lower in patients compared to healthy donors (0.4 vs 41.6 U/ml respectively; p < 0.0001) (Fig. 2A,C).The median titre amongst patients with a positive result was 3.96 (10.5-fold lower than healthy donors) whilst the median titre response of those who responded to the first vaccine but had no evidence of previous infection was 2.49 (16-fold lower than healthy donors). Several studies have now shown that people who have had a previous natural SARS-CoV-2 infection exhibit particularly strong immune responses following Covid-19 vaccination.Previous infection can be determined by the presence of nucleocapsidspecific antibodies and these were detected in 5 donors with CLL.Within this group, median antibody titres within S1 samples were boosted by a remarkable 21,450-fold to reach a median value of 8580 U/ml (naturally infected median 8580 U/ml vs no previous infection 0.4 U/ml; p < 0.0001).Of note these values are comparable to the levels of 10,700 U/ml in samples from healthy donors with previous infection after one vaccine (p > 0.999). Given the clinical heterogeneity amongst patients with CLL we next assessed antibody responses in relation to clinical status and management.Patients were divided into 5 groups: 'watch and wait'; previously completed chemo-immunotherapy (median 68 months (IQR 35-115) since last treatment); current treatment with BTK inhibitor; current treatment with venetoclax; and 'plans to start treatment imminently'.Analysis of serum samples showed that responses were low in all five groups and, although numbers within the subgroups were small, patient responses were particularly suppressed amongst those on current therapy or those who were due to start therapy in the near future (Fig. 2B). Following analysis of the serum samples we went on to determine the spike-specific antibody response from dried blood spot samples (DBS) using a spike-specific ELISA (The Binding Site (TBS)).These DBS1 samples were available in 267 patients and compared to values from 93 age-matched healthy donors following single vaccination.Analysis of paired samples showed a strong correlation between the spike-specific antibody response detected by serum and DBS within the patient group (r = 0.65; p < 0.0001) (Supplementary Fig. 1A) and confirms previous work within healthy donors [15]. Antibody responses were detectable in 63 of the 267 DBS1 patient samples (24%) which compares to the value of 34% from the serum analysis.Positive responses were detectable in 71% of the control group and as such were somewhat lower than responses obtained from analysis of serum samples (Fig. 2D,F).The magnitude of antibody response from DBS eluates was also lower within the patient group compared to controls (median ratio to calibrator of 0.5 vs 1; p < 0.0001) (Fig. 2D).Amongst the 24% of patients where a positive antibody response was seen, a similar median ratio was observed in patients with CLL and healthy donors (CLL: 1.76 vs HD 1.71).Analysis of DBS1 eluate samples showed low responses across all patient groups.Comparison between subgroups showed that patients on BTKi therapy were less likely to develop a positive antibody response compared to those on watch and wait (p = 0.0056) (Fig. 2E).No difference was observed between patients who received the BNT162b2 and ChAdOx1 vaccines (Supplementary Fig. 1B). ", "section_name": "Patient characteristics", "section_num": null }, { "section_content": "We next went on to assess antibody responses at 2-3 weeks following the second vaccine.At the current time, due to widespread adoption of the 'extended interval' vaccine regimen in the UK, samples are available on only 12 serum and 55 DBS samples (termed S2 and DBS2 samples, respectively).Spikespecific antibody responses were identified in 9 of the 12 serum samples from the patient group (75%) compared to a 100% response rate in healthy age-matched controls (n = 59) (Fig. 3C).The titre of this anti-spike antibody response was 74-fold lower in patients with CLL compared to healthy age-matched donors without previous infection (n = 59) (53 U/ml vs 3900 U/ml; p < 0.0001) (Fig. 3A,C).Amongst the 75% of patients with a positive antibody response, the median response was 102 U/ml.Eight of these patients were on 'watch and wait' management (Fig. 3B) and no patients in the group had serological evidence of previous natural SARS-CoV-2 infection. Spike-specific antibody responses were then assessed in the 55 DBS2 samples.Antibodies were detectable in 71% of patients (39/55) compared to 97% (36/37) of samples from healthy donors (n = 37) (Fig. 3F).Antibody levels, as assessed by the DBS2 ratio, were also significantly lower amongst the 55 patients (2.9 vs 4.5 healthy donors; (p = 0.0004; Fig. 3D), whilst those with a detectable antibody response had a median ratio of 3.86.Analysis of responses in relation to clinical status showed lower antibody levels in patients on BTKi therapy (Figs.3E and4B). Paired samples after the first and second vaccine were available for 27 donors and revealed similar fold-increment in antibody levels between timepoints within both the patient group and 19 agematched healthy donors (Fig. 4A).As such these data indicate that patients with CLL get an equivalent proportionate antibody response after the second vaccine although values both before and after this vaccine remain lower than those in the control group. Total serum immunoglobulin levels were also determined on DBS2 samples for subsequent assessment of their relationship to spike-specific antibody response.Seventeen of the 55 patients (35%) were found to be IgG deficient, 19 (32%) were IgA deficient and 16 (30%) were IgM deficient (Fig. 4C).A combined deficiency of IgG and IgA was present in 10 (18%) patients (Fig. 4D). ", "section_name": "Antibody responses improve after second vaccination but remain low compared to age-matched controls", "section_num": null }, { "section_content": "The relative importance of individual clinical and laboratory variables on the probability of developing a positive spike-specific antibody response after second vaccination were then assessed in univariate analysis against values obtained from DBS2 samples (Table 2). The age of the patient and the duration of time since diagnosis were not associated with the probability of generating a positive antibody response (Supplementary Fig. 2).Antibody response rates were lower in patients who reported a history of severe infection but this did not reach statistical significance.Serum concentrations of IgG, IgA or IgM all showed positive correlations with antibody response but this remained significant only for IgA in multivariate analysis (IgA: OR 9.1; 2-42, p = 0.005).Current therapy with BTK inhibitors was associated with markedly reduced likelihood of a positive response to vaccination and remained significant in multivariate analysis (OR: 0.05; 95% CI: 0.004-0.58,p = 0.016). ", "section_name": "Determinants of response to second vaccine", "section_num": null }, { "section_content": "SARS-CoV-2 vaccines have proven highly effective in protection against severe Covid-19 but there remains considerable concern about their efficacy in patients with immune suppression [16]. Here we show that immune responses elicited after the first and second vaccine are substantially reduced in patients with chronic lymphocytic leukaemia.These findings raise a number of questions in relation to optimisation of vaccine protection in this vulnerable cohort. The median age of our cohort was 69 years and as such it would be expected that immune senescence will play a role in vaccine response [17].Importantly we were able to compare the patient responses with a large age-matched control group.Antibody responses within the patient group after one vaccination were low and detectable in only 34% compared to 94% of age-matched controls.The antibody titre was also over 100-fold lower at this time.These results are comparable with the 18% response rate in a haematological cancer cohort that included 11 CLL patients [16]. Interestingly, 5 of the 86 donors for which serological samples were available showed the presence of nucleocapsid-specific antibodies indicating previous natural infection and in this group the antibody response after first vaccine was remarkably high and directly comparable to those seen in previously infected healthy donors after vaccination.Indeed, antibody levels were markedly higher than those seen in previously uninfected patients after two Covid-19 vaccines.As such, immune memory after natural infection appears able to overcome the impaired antibody response to the Fig. 3 Antibody responses in patients with CLL following second Covid-19 vaccine.A Antibody responses to SARS-CoV-2 Spike following second vaccine in sera from healthy donors (HD) and patients with CLL, as measured by Roche assay.No donors had evidence of previous exposure (cut off for positivity at 0.8 indicated by dotted line).B Dot plot of antibody responses to SARS-CoV-2 Spike in patient with CLL following second vaccine, by management stage shown, (W+W watch and wait; PCi previous chemo-immunotherapy but not on active therapy) as measured by Roche.C Bar chart to show the percentage response after second vaccine in HD and CLL measured by Roche.D Antibody responses to SARS-CoV-2 spike following second vaccine in healthy donors (HD) and patients with CLL, as measured by TBS assay (cut off for positivity indicated by the dotted line at a ratio of 1).E Antibody responses to SARS-CoV-2 Spike in patient with CLL following second vaccine, by management stage, DBS testing and analysed by TBS assay (Kruskal-Wallis p < 0.0045 and Dunn's analysis for BTKi therapy and W+W p = 0.03).Watch and Wait (W+W); Previous Chemo-immunotherapy but not on active therapy (pCI); Bruton Tyrosine Kinase therapy (BTKi); Treatment planned (TP).F Bar chart to show the percentage response after second vaccine in HD and CLL measured by TBS assay using DBS eluates.initial vaccine in the patient group.Similar findings in relation to the 'vaccine-priming' effect of previous natural infection have previously been reported [15,18,19].Of interest, only 2 of these 5 patients reported symptoms compatible with SARS-CoV-2 infection over the last 12 months suggesting that asymptomatic infection occurs amongst patients with CLL and that mortality rates may have therefore been overestimated [3,5].One limitation to this finding may be that some patients with CLL who had previous natural infection may lack a nucleocapsid-specific antibody response in the convalescent serum due to antibody waning, secondary immunodeficiency or hypogammaglobulinaemia. Encouragingly, antibody responses increased after the second vaccine and were positive in 75% of serum samples and 71% of DBS eluates at this time point.These values are somewhat higher than the response rate of 40% in a recent study of 167 patients after the second BNT162b2 vaccine [13].Further assessment of donors within our study at this time point is ongoing as more samples are collected.Of note, most patients in this study were studied after extended interval vaccination and this may potentially serve to boost antibody responses following the second vaccine [7].Our study has found no difference in antibody levels following the BNT162b2 or ChAdOx1 vaccines to date. Of interest, spike-specific antibody levels within patients after second vaccination were broadly comparable to those seen in the control group after a single vaccine which is noteworthy given the clinical protection demonstrated following a single vaccine within the general population [20]. We were further interested to see how vaccine responses varied in relation to clinical and laboratory features of individual patients.Patient age, or time since original diagnosis, did not impact significantly on vaccine response.In contrast the serum level of immunoglobulin was a notable determinant, with higher levels of all three isotypes associated with improved antibody response rate in univariate analysis.IgA deficiency emerged as the most significant predictor of poor vaccine response and normal levels were associated with a 10-fold increase in the probability of a positive response after second vaccination.IgA is the first of the immunoglobulin classes to diminish in patients with CLL [21] and this is reflected in this cohort where 32% of participants were deficient in IgA.IgA levels at diagnosis have also been shown to predict infection risk [22] and IgA deficiency has been associated with poor responses to pneumococcal polysaccharide vaccines [23].Herishanu et al. also found that having normal immunoglobulins predicted better response rates to the BNT162b2 vaccination [13]. Bruton tyrosine kinase inhibitor therapy was a strong and independent predictor of negative antibody response after the second vaccine.BTKi therapy has transformed the management of patients with CLL over the past decade but its impact on vaccine response has been previously reported and is unsurprising given the pivotal role of BTK in B-cell activation [8,11].It would appear advisable that patients who are being considered for BTKi therapy should receive their Covid-19 vaccination prior to therapy where possible.However, we also observed suboptimal vaccine responses in untreated patients who were planning to start therapy in the near future and this is likely to reflect the immunosuppressive impact of active disease.In contrast, positive antibody responses were observed in 83% of patients who were on 'watch and wait' management and this was particularly true for those with normal serum immunoglobulin levels. Relatively few patients in our cohort were in remission from previous chemotherapy but it appears that antibody responses in this group are also more robust and this would be compatible with previously published work [13].Other limitations of our work include patient self-reporting for infection history and previous COVID-19 infection, a lack of nucleocapsid-specific antibody assessment in the DBS samples, inability to link to current haematological parameters, and the relatively small number of samples after the second vaccine as a result of the extended interval programme. Our findings raise questions regarding optimal vaccination policy for patients with CLL and the potential need for additional management.The great majority of the UK population is receiving the Covid-19 vaccine with an 'extended interval' of 10-12 weeks between doses.As such, in this interim report the majority of samples were obtained after the first vaccine but before the second dose and suggest that with CLL should be considered for early delivery of the second vaccine.However, antibody responses may remain suboptimal even after two vaccines.One option might be to consider a third 'booster' vaccine and this is being implemented in some countries for patients in other risk groups.At this stage it is not clear if a third vaccine will indeed act to further boost antibody responses although the substantial increment after the second boost and the high antibody titres observed following natural infection suggest that this may be possible.It is possible that vaccination may serve to provide sufficient 'immune-priming' to protect against severe disease from subsequent infection even in the absence of a measurable spike-specific antibody response.Indeed, we have not yet assessed the profile of spike-specific cellular immunity following vaccination and, although cellular immune responses are also typically suboptimal in patients with CLL, these are likely to provide some protection from severe disease [24].However, antibody levels are emerging as a correlate of immune protection [20,25] and as such the administration of prophylactic SARS-CoV-2-specific antibodies to patients at the greatest clinical risk could be valuable. In conclusion we show that antibody responses following Covid-19 vaccination are reduced in patients with CLL, with patients who are IgA-deficient or on BTKi therapy at particular risk for failure to develop a response.It is now critical that the clinical efficacy of vaccination is determined in this patient group using data linkage from large population datasets and this assessment is underway in many countries.This information, together with assessment of immune correlates, will be important to guide the ongoing requirement for behavioural adjustments such as social distancing. ", "section_name": "DISCUSSION", "section_num": null } ]	[ { "section_content": "We would like to thank the patients for their time and willingness to take part and also the charities for their support, including CLLSA, Leukaemia Care and Blood ", "section_name": "ACKNOWLEDGEMENTS", "section_num": null }, { "section_content": "HP, PM wrote the manuscript.HP, RB and PM designed the study and HP, RB, GM, NW, SP, GP, TM, HR and PH recruited participants.JZ, CS, MA, AO, SF and AR performed the experiments.HP, GM, SD and JZ analysed the data.All authors commented on the manuscript. Ethical approval was obtained from North West Preston Research Ethics Committee with favourable outcome.Work was performed under the CIA UPH IRAS approval. The authors declare no competing interests. The online version contains supplementary material available at https://doi.org/10.1038/s41408-021-00528-x. Correspondence and requests for materials should be addressed to P.M. Reprints and permission information is available at http://www.nature.com/reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "HP, PM wrote the manuscript.HP, RB and PM designed the study and HP, RB, GM, NW, SP, GP, TM, HR and PH recruited participants.JZ, CS, MA, AO, SF and AR performed the experiments.HP, GM, SD and JZ analysed the data.All authors commented on the manuscript. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "Ethical approval was obtained from North West Preston Research Ethics Committee with favourable outcome.Work was performed under the CIA UPH IRAS approval. ", "section_name": "ETHICAL APPROVAL", "section_num": null }, { "section_content": "The authors declare no competing interests. ", "section_name": "COMPETING INTERESTS", "section_num": null }, { "section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41408-021-00528-x. Correspondence and requests for materials should be addressed to P.M. Reprints and permission information is available at http://www.nature.com/reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "ADDITIONAL INFORMATION Supplementary information", "section_num": null } ]
10.1371/journal.pone.0050826	Hepatitis C Virus Mediated Changes in miRNA-449a Modulates Inflammatory Biomarker YKL40 through Components of the NOTCH Signaling Pathway	Liver disease due to hepatitis C virus (HCV) infection is an important health problem worldwide. HCV induced changes in microRNAs (miRNA) are shown to mediate inflammation leading to liver fibrosis. Gene expression analyses identified dysregulation of miRNA-449a in HCV patients but not in alcoholic and non-alcoholic liver diseases. By sequence analysis of the promoter for YKL40, an inflammatory marker upregulated in patients with chronic liver diseases with fibrosis, adjacent binding sites for nuclear factor of Kappa B/P65 and CCAAT/enhancer-binding protein alpha (CEBPα) were identified. P65 interacted with CEBPα to co-operatively activate YKL40 expression through sequence specific DNA binding. In vitro analysis demonstrated that tumor necrosis factor alpha (TNFα) mediated YKL40 expression is regulated by miRNA-449a and its target NOTCH1 in human hepatocytes.NOTCH1 facilitated nuclear localization of P65 in response to TNFα. Further, HCV patients demonstrated upregulation of NOTCH1 along with downregulation of miRNA-449a. Taken together it is demonstrated that miRNA-449a plays an important role in modulating expression of YKL40 through targeting the components of the NOTCH signaling pathway following HCV infection. Therefore, defining transcriptional regulatory mechanisms which control inflammatory responses and fibrosis will be important towards developing strategies to prevent hepatic fibrosis especially following HCV recurrence in liver transplant recipients.	[ { "section_content": "Liver diseases resulting from hepatitis C virus (HCV) infection is a major health issue worldwide as well as the United States [1,2].It is estimated that about 4 million people are infected with HCV in the United States and about 300 million worldwide [1].The natural history of HCV infection in the liver is characterized by slow progression to fibrosis and cirrhosis, end-stage liver diseases, and high risk of developing hepatocellular carcinoma (HCC) [3].YKL40 (CHI3L1) is a member of the ''mammalian chitinaselike proteins,'' secreted by activated macrophages and neutrophils during inflammation in various tissues including liver, smooth muscle and cancer cells [4].YKL40 is elevated in patients with chronic liver diseases that are characterized by inflammation and increased extra-cellular remodeling [5,6].Although increased levels of YKL40 have been shown to be induced by tumor necrosis factor alpha (TNFa), the molecular mechanisms are not clearly identified [7].TNFa, an inflammatory cytokine regulates gene expression in the nuclear factor of Kappa B (NFKB) signaling pathway [8].Components of the mammalian NFKB family of transcription factors includes NFKB1 (P105/P50), NFKB2 (P100/ P52), RelA (P65), RelB and c-Rel [9].The NFKB component P65 is a multimeric DNA binding transcription factor involved in inflammatory and immune disorders especially autoimmune diseases and cancer [10].NOTCH1 is one of the upstream regulator of NFKB complex and downregulation of NOTCH1 impairs its function [11,12].It has been shown that NOTCH1 and TNFa regulate nuclear retention of NFKB [13,14].CCAAT/ enhancer-binding protein alpha (CEBPa) is a homodimeric DNA binding bZIP transcription factor that controls cell proliferation and differentiation [15].CEBPa is differentially regulated in cases of HCC and targets expression of a wide range of genes and microRNAs (miRNA) involved in liver diseases [16,17]. miRNAs have been shown to play an important role in immune evasion, regulation of cell cycle and in cancer progression [18,19,20].HCV infection results in modulation of miRNA particularly those that control viral particle entry and propagation, thus playing an important role in host immune evasion [21].In this study we defined the molecular mechanisms of YKL40 expression that involves HCV induced miRNA modulation and regulation by novel pathways including NOTCH1, NFKB and ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "Liver biopsies were obtained from 10 chronic HCV patients, 10 alcoholic hepatitis patients, 10 non-alcoholic steatohepatitis patients and 10 normal donor livers (control) at the time of transplantation at Washington University Medical Center/Barnes-Jewish Hospital (Table 1).Patients with hepatitis B virus and/or HIV were excluded from the study.All of the human studies were approved by the human research protection committee at Washington University (protocol 201104075) and patients were enrolled after written informed consent was obtained. ", "section_name": "Patients", "section_num": null }, { "section_content": "For YKL40 luciferase constructs, the promoter regions were amplified from human genomic DNA (Zyagen, CA) by PCR using iProof High-Fidelity DNA Polymerase (Biorad, CA).PCR products were subcloned into pGL4.11vector (Promega, WI) upstream of a luciferase gene using the NheI/EcoRV restriction sites.P65 and CEBPa were amplified from a human cDNA library (Stratagene, CA) and subcloned into pcDNA using the HindIII/ Not1 and HindIII/BamH1 restriction sites, respectively.Hsa-miRNA-449a (SC400399) and control constructs were purchased from Origene, MD.NOTCH1 (sc-36095), P65 (sc-29410) and control siRNA (sc-37007) were purchased from Santacruz Biotechnology, CA.Computational analysis of the promoter bound transcription factors was done using the Transcription Element Search System http://www.cbil.upenn.edu/cgi-bin/tess/tess. miRNA target analysis was done using http://www.targetscan.org. ", "section_name": "Plasmids and Constructs", "section_num": null }, { "section_content": "Total RNA was isolated from the liver biopsies or hepatocytes using the RNAaqueous kit (Ambion, NY).Expression level of miRNA-449a was determined using the TaqManH MicroRNA assays and TaqManH Universal Master Mix II (Life technologies, NY) using predesigned primers.Quantitative PCR (qPCR) to analyze YKL40 and NOTCH1 was performed using a BioRad Real-Time PCR System with cycling conditions of 95uC for 10 min followed by 95uC for 15 sec and 60uC for 60 sec for a total of 40 cycles.Each TaqMan assay was run in triplicate.The DDCt value was calculated by normalizing the threshold (CT) values with GAPDH expression and respective gene expression in controls. ", "section_name": "miRNA and mRNA Expression Analysis", "section_num": null }, { "section_content": "Primary human hepatocytes were purchased from Life Technologies, New York and grown in 24 well plates in Williams Medium E supplemented with 5% FCS, 100 units/mL penicillin, 100 units/ml Amphotericin, 0.1% Albumin, 300 nM insulin, 2 mM L-glutamine and 0.1 nM Hydrocortisone.HEPG2 cells were grown in RPMI with 10% FBS, 1 mM sodium-pyruvate, 10 mM HEPES, 2 mM L-glutamine, and 100 units/mL penicillin/streptomycin. 20 ng/ml of TNFa (Sigma, St. Louis, MO) was added for 6 hours wherever indicated.The optimal concentration of TNFa was determined by dose-dependent analyses. For transfection of hepatocytes and HEPG2 cells (ATCC), 0.2610 5 cells were seeded into each well of a 24 well plate and grown for 24-48 hours.On the day of transfection, medium was changed and 500 ng of DNA was transfected using Lipofectamine TM LTX and Plus Reagent (Invitrogen, NY).For siRNA delivery 0.1610 5 cells were grown in each well of a 24 well plate for 24-48 hours in antibiotic free medium and 80 pico moles of siRNA were transfected using Lipofectamine TM RNAiMAX (Invitrogen).Cells were harvested 48 hours post transfection and efficiency was measured by qPCR, immunostaining and western blot. ", "section_name": "Primary Hepatocytes and HEPG2 Cell Line Transfection", "section_num": null }, { "section_content": "For localization of YKL40, NOTCH1 and P65, 50,000 HEPG2 cells were grown on coverslips in 24 well plates.Immunostaining was done as described before [22].Briefly, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100.2% normal goat serum in DPBS with 1% BSA, 0.1% Tween 20 was used for blocking and washing.Primary antibodies for western blot and Immunofluorescence used were goat anti-YKL40 (sc-31722), rabbit anti-NOTCH1 (sc-9170), mouse anti-CEBPa (sc-166258) and rabbit anti-P65 (sc-109).The secondary antibodies used were FITC-conjugated anti-mouse IgG (sc-2010), Rhodamine-conjugated anti-goat IgG (sc-3945) and Rhodamine-conjugated anti-rabbit IgG (sc-2492).The images were captured using an Eclipse 80i fluorescent microscope (Nikon, NY) and processed using Metamorph version 6.3r2 software (Molecular Devices, CA).Extraction of the nuclear and cytoplasmic fractions from the hepatocytes (1610 6 cells) was done using NE-PERH Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, IL). ", "section_name": "Western Blot and Immunofluorescence Microscopy", "section_num": null }, { "section_content": "Human primary hepatocytes (1610 5 ) were transfected in 24 well plates as mentioned earlier with 1 mg pGL4.11luciferase reporter vector or pGL4.11driven by the YKL40 promoter or deletion constructs.For miRNA regulation studies the reporter construct was transfected in combination with 1 mg of either control vector or vector expressing miRNA-449 precursor.For NOTCH1 regulation studies the reporter construct was transfected in combination with 80 picomoles of either non specific siRNA or siRNA specific for NOTCH1.For transcription factor studies the reporter constructs were transfected in combination with 2 mg of empty pcDNA3 vector, or pcDNA3 expressing P65 or CEBPa or both.20 ng/ml of TNFa was added to the medium 6 hours before harvesting.To control for efficiency of transfection, 0.1 mg of pRL-TK (Promega, Madison, WI), which expresses Renilla luciferase was included.Luciferase activity was measured 48 h after electroporation using the Dual Luciferase Reporter Assay System (Promega, Madison, WI) and the results were normalized to Renilla luciferase. ", "section_name": "Luciferase Assay", "section_num": null }, { "section_content": "Immunoprecipitation of P65 with CEBPa or the reverse immunoprecipiation in TNFa treated hepatocytes (1610 6 ) were carried out as described by Sarma et al [22].Briefly, cells were washed with DPBS, and lysed with 0.5 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 0.4 M NaCl, 1% Nonidet P-40, 0.4% Triton X-100, 0.2% sodium deoxycholate, 1 mM EDTA, protease inhibitors (PI), 1 mM PMSF).Diluted with 0.5 ml buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, PI, 1 mM PMSF and centrifuged at 17,0006g for 30 min. 1 mg normal mouse/ rabbit IgG or mouse anti-CEBPa or rabbit anti-P65 was used to immunoprecipitate the complexes from the supernatant.After overnight incubation with the antibodies 30 ml of Protein G beads were added to lysates and incubated for another 1 hour.Beads were washed with 700 ml of wash buffer (10 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 0.5% Nonidet P-40, 0.2% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA, PI, 1 mM PMSF) 3 min each for 5 times and once with cold DPBS by centrifugation at 1,8006g for 3 min at 4uC.Immunocomplexes were eluted by boiling with 30 ml of 2X SDS buffer (0.1 M Tris-HCl, pH 6.8, 3.5% SDS, 10% glycerol, 2 mM DTT, 0.004% bromphenol blue) for 10 min and subjected to SDS-PAGE (4-20% gel).P65 or CEBPa were detected with a rabbit anti-P65 or a mouse anti-CEBPa respectively. ", "section_name": "Co-immunoprecipitation", "section_num": null }, { "section_content": "Chromatin Immunoprecipitation was carried out with ChIP-IT TM Express (Active Motif, Carlsbad, CA).Briefly, hepatocytes (1610 6 ) were crosslinked with 1% formaldehyde and quenched with 0.375 M glycine.Nuclei were isolated and sonicated in 350 ml of shearing buffer to prepare chromatin extracts.1 mg of antibodies for control IgG, P65 or CEBPa were added to 60 ml of sheared chromatin along with Protein G Magnetic Beads.Antibody-lysate mix was washed and DNA was eluted according to the instructions.YKL40 promoter regions were amplified by PCR. ", "section_name": "Chromatin Immunoprecipitation", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "A genomewide miRNA analysis in liver biopsies obtained from chronic HCV infected patients demonstrated a distinct expression profile when compared to the normal liver.Particularly, a significant downregulation of microRNA-449a was observed in the HCV infected livers. To analyze the specific role of miRNA-449a following HCV infection, biopsies were obtained from 10 chronic HCV patients, 10 alcoholic hepatitis patients, 10 non-alcoholic Steatohepatitis (NASH) patients and 10 control normal donor livers at the time of liver transplant.Total RNA was isolated from the liver biopsies and expression level of miRNA-449a was determined by qPCR using specific primers and the results were normalized to GAPDH expression.Expression analysis demonstrated that miRNA-449a is downregulated more than two fold in livers obtained from HCV patients whereas no significant differences in the expression was observed in alcoholic hepatitis patients, NASH patients and 1A).This suggests that miRNA-449a is specifically downreguated in patients with liver diseases following HCV infection. ", "section_name": "miRNA-449a is Downregulated in HCV Patients", "section_num": null }, { "section_content": "To determine the expression level of YKL40 in liver diseases, biopsies were obtained from 10 chronic HCV patients, 10 alcoholic hepatitis patients, 10 NASH patients and 10 control normal donor livers at the time of liver transplant.Total RNA was isolated from the liver biopsies and expression level of YKL40 was determined by qPCR using specific primers and the results were normalized to GAPDH expression.Expression analysis showed that YKL40 to be upregulated in alcoholic hepatitis patients (2.4 fold), NASH patients (2.9 fold) and HCV patients (2.6 fold) compared to normals (Figure 1B).These results demonstrate that YKL40 is elevated in patients with chronic liver diseases which are accompanied by inflammation. ", "section_name": "YKL40 is Upregulated in HCV Patients with Fibrosis", "section_num": null }, { "section_content": "We and others have shown that YKL40 expression is elevated in patients with chronic liver diseases accompanied by inflammation [5,6].In vitro studies have shown that inflammatory cytokines such as TNFa have the ability to induce expression of YKL40 [7].To study the molecular mechanisms of TNFa mediated regulation of YKL40, 23000 base pairs (bp) of the human YKL40 promoter was cloned upstream of a luciferase reporter gene and the construct was introduced into human hepatocytes.The cells were treated either with or without 20 ng/ml TNFa for 6 hours and luciferase activity was measured.More than four fold increase in the YKL40 promoter activity was observed in cells treated with TNFa compared to untreated (Figure 2A, upper panel).Further, western blot analysis showed increased expression of YKL40 in TNFa treated cells (Figure 2A, lower panel).This reporter gene analysis suggests that TNFa regulates the expression of YKL40 through modulation of upstream transcriptional complexes that interact with the YKL40 promoter. To further demonstrate that TNFa induces expression of YKL40, human HEPG2 cells were cultured and treated with TNFa.Immunostaining of the HEPG2 cells with anti-YKL40 showed a more than 2 fold increase in expression of YKL40 in the cells treated with TNFa compared to untreated cells (Figures 2B and2C). ", "section_name": "TNFa Regulates the Expression of YKL40 at the Transcriptional Level", "section_num": null }, { "section_content": "Since TNFa regulated the expression of YKL40 at the transcriptional level we hypothesized the possible interaction of TNFa with upstream regulatory complexes of the YKL40 gene.To identify the essential regions for TNFa mediated expression deletion mutants of the YKL40 promoter regions were cloned upstream of a luciferase reporter gene.Sequential deletion mutants of the YKL40 promoter region (Figure 2D, black filled bars on the left) were introduced into hepatocytes.The cells were treated with or without TNFa and the luciferase activity was measured.Significant increases (.10 fold) in the expression from the YKL40 promoter deletion constructs were observed in the cells treated with TNFa compared to untreated (Figure 2D).However, deletion of the 21000 to 2500 bp region of the YKL40 promoter impaired TNFa mediated transcriptional induction.This indicates that this region (21000 to 2500 bp) contains binding sites for transcriptional regulatory elements on the YKL40 promoter. ", "section_name": "Reporter Analysis Identified Essential Regions in the YKL40 Promoter Required for TNFa Regulated Expression of YKL40", "section_num": null }, { "section_content": "In order to identify transcription factors that regulate the expression of YKL40 a computational analysis of 24000 bp upstream of the open reading frame was done using the transcription element search system.The program predicted consensus binding sites in the DNA for several transcription factors that included NFKB subunit P65 and CEBPa (Figure 3A).The software predicted a consensus binding site for p65 (GGAATTTCC) at 2578 bp position at the promoter.Similarly several DNA binding sites for CEBPa (CCAAT) was also predicted throughout the YKL40 promoter.Most of the CEBPa binding sites were concentrated in the 23000 to 22000 bp region of the promoter.Interestingly, two CEBPa binding sites were identified in close proximity to the P65 DNA binding site on the promoter (Figure 3A) suggesting that these two transcription factors may bind to the YKL40 promoter by forming a transcriptional regulatory complex. ", "section_name": "Computational Prediction of Transcription Factors Regulating YKL40 Expression", "section_num": null }, { "section_content": "Computational analysis predicted a putative binding site for NFKB subunit P65 at 2578 bp position of the human YKL40 promoter.Further the YKL40 promoter analysis using the deletion constructs identified 21000 to 2500 bp region to be essential for TNFa mediated induction of YKL40 in hepatocytes.To test whether P65 binding plays a role in TNFa mediated upregulation of YKL40 promoter a 2578 bp wildtype luciferase reporter construct, a 2578 bp construct with point mutations in the P65 binding site (wildtype: GGAATTTCC, point mutation: AGCT-TATCA) and a 2500 bp construct with P65 binding site deleted were prepared.Hepatocytes were transfected with these three reporter constructs and cells were treated either with or without TNFa and Luciferase activity was measured.As expected the wildtype 2578 bp promoter construct with an intact P65 binding site showed two fold increase in transcriptional activity in the presence of TNFa (Figure 3B).However, point mutation in the P65 binding site or deletion of the P65 binding site (2500 bp) completely abolished TNFa mediated induction of YKL40 (Figure 3B).Further, we demonstrated that siRNA mediated knockdown of P65 resulted in impairment of transcriptional activation by the YKL40 promoter in TNFa treated cells. ", "section_name": "Mutation of NFKB (P65) Binding Site Inhibits TNFa Mediated YKL40 Induction", "section_num": null }, { "section_content": "Computational analysis identified CEBPa as a putative DNA binding factor for transcriptional regulation of YKL40.To test whether CEBPa regulates YKL40 expression an empty vector or numbers represent fold-change over the 2578 wildtype construct without TNFa treatment (average of three independent experiments); error bars represent SD.The P65 binding site mutation is shown in the lower panel.The '*' represents p value ,0.05 obtained by a two-tailed t test.C. CEBPa in an upstream transcription factor to activate YKL40 expression.Hepatocytes were transfected with luciferase reporters driven by deletion constructs of YKL40 promoter (23000 bp, 22000 bp, 21000 bp, 2500 bp, filled black bars on left) along with an empty vector or vector expressing CEBPa and treated with TNFa.Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control.The numbers represent fold-change over the control empty luciferase vector (average of three independent experiments); error bars represent SD. doi:10.1371/journal.pone.0050826.g003a vector expressing CEBPa was overexpressed in hepatocytes along with deletion mutants of the YKL40 promoter cloned upstream of a luciferase reporter gene.The deletion mutants included 23000 bp, 22000 bp, 21000 bp and 2500 bp of the YKL40 promoter region (Figure 3C, black filled bars on the left) and the luciferase activity was measured after the cells were treated with TNFa for 6 hours.Significant increases (.9 fold) in the YKL40 expression from the 23000 bp promoter construct was noted when CEBPa was overexpressed compared to control empty vector (Figure 3C).However, deletion of the 23000 bp to 22000 bp region of the YKL40 promoter impaired CEBPa mediated transcriptional induction of YKL40 as that region contained several putative CEBPa binding sites as shown on our computational analysis (Figure 3A). ", "section_name": "CEBPa in an Upstream Transcription Factor which Activate YKL40 Expression", "section_num": null }, { "section_content": "Reporter analysis demonstrated that both P65 and CEBPa regulate expression from the YKL40 promoter.To test whether P65 and CEBPa interact and bind to adjacent consensus sites present on the YKL40 promoter a chromatin immunoprecipitation analysis was done.TNFa treated hepatocytes were fixed with formaldehyde and chromatin extracts were prepared.The DNAprotein complexes were immunoprecipitated with anti-P65 or anti-CEBPa or isotype control IgGs.Chromatin fragments were isolated from the immunoprecipitated DNA-protein complexes and subjected to PCR amplification using primers specific for YKL40 promoter regions as shown in Figure 4A.The PCR products were resolved on agarose gel.Chromatin immunoprecipitation analysis showed that both P65 and CEBPa bind to the YKL40 promoter (Figure 4B).P65 binds to the 2300 bp to 2700 bp region of the YKL40 whereas no binding was observed with other regions of the promoter.This region encompasses the P65 binding site at 2578 bp position.CEPBa showed binding to several regions of the YKL40 promoter where maximum band intensity was seen at 21900 bp to 22300 bp region that encompasses most of the CEBPa binding sites.Immunoprecipitation with isotype IgGs did not enrich any of these YKL40 promoter regions demonstrating the specificity for P65 or CEBPa.No amplification of DNA was observed in PCR reactions performed with primers specific for ACTIN promoter. Binding of both CEBPa and P65 to the YKL40 promoter in adjacent DNA binding sites suggests the possibility that they interact with each other.To test this, crude lysates from TNFa treated hepatocytes were prepared and subjected to immunoprecipitation with either isotype control IgG or anti-P65 followed by immunoblotting with anti-CEBPa.CEBPa was co-immunoprecipitated with endogenous P65 whereas no CEBPa was observed with the control IgG (Figure 4C).To confirm the interaction a reverse co-immunoprecipitaion was done by immunoprecipitation with either isotype control IgG or anti-CEBPa followed by immunoblotting with anti-P65 (Figure S1).To further demonstrate the interaction between P65 and CEBPa, HEPG2 cells were treated with or without TNFa and co-immunostained with anti-CEBPa and anti-P65.CEBPa primarily localized to the nucleus in both TNFa treated and untreated cells.P65 remained exclusively cytoplasmic in the untreated cells with little to no nuclear localization.In the TNFa treated cells, a significant amount of P65 translocated into the nucleus and co-localized with CEBPa (Figure 4D). Immunoprecipitation and co-localization analyses indicated that both P65 and CEBPa interact with each other and bind to their consensus sites on the YKL40 promoter.To determine their role in transcriptional regulation from the YKL40 promoter, hepatocytes were co-transfected with a luciferase reporter construct driven by the YKL40 promoter (23000 bp) with P65 or CEBPa or both.Overexpression of both P65 and CEBPa resulted in significant increase in transcriptional activation of the reporter construct compared to either alone (Figure 4E, upper panel).Immunoblotting of the cell lysates with anti-P65 and anti-CEBPa confirmed the elevated expression levels of these factors compared to endogenous levels (Figure 4E, lower panel).This demonstrates that CEBPa cooperates with NFKB to regulate expression from the YKL40 promoter. ", "section_name": "CEBPa Interacts with NFKB/P65 to Bind YKL40 Promoter and Cooperates to Activate Transcription in Hepatocytes", "section_num": null }, { "section_content": "Results presented clearly demonstrates that TNFa mediated regulation of YKL40 is dependent on the NFKB subunit P65.To demonstrate nuclear translocation of P65 in response to TNFa in hepatocytes, cells were treated with or without TNFa and cytoplasmic and nuclear extracts were prepared.Whole cell lysates, the cytoplasmic fraction and the nuclear fraction were subjected to immunoblotting with anti-P65.The expression of P65 was not affected by TNFa as no difference was observed in the whole cell lysate (Figure 5A, left panel).However, significant cytoplasmic exclusion and nuclear enrichment of P65 was observed in TNFa treated cells compared to untreated cells.One of the essential upstream regulators of NFKB complex is NOTCH1 [11,12].To analyze the role of NOTCH1, an upstream factor required for TNFa mediated nuclear localization of P65, and its functionality in facilitating downstream gene regulation, hepatocytes were transfected either with scrambled siRNA or siRNA specific for human NOTCH1 and subjected to TNFa treatment.The siRNA mediated knockdown of NOTCH1 was confirmed by immunoblotting with anti-Notch1 (Figure S2A).Immunoblot using anti-P65 demonstrated that knockdown of NOTCH1 resulted in impairment of TNFa mediated cytoplasmic exclusion and nuclear translocation of P65 (Figure 5A, right panel). To further demonstrate that NOTCH1 is required for TNFa mediated translocation of P65, HEPG2 cells were treated with or Chromatin was immunoprecipitated with anti-P65 or anti-CEBPa or isotype control IgG from hepatocytes.Segments of the YKL40 promoter (indicated in 4A) were amplified by PCR.The first three lanes show immunoprecipitated chromatin (IP) and the fourth lane show input chromatin (Input).ACTIN promoter amplification is shown as the negative control.C. Co-immunoprecipitation of P65 with CEBPa in hepatocytes.Whole cell lysates were subjected to immunoprecipitation with either rabbit IgG or anti-P65.CEBPa in the cell lysates (Input) and immunoprecipitated complexes (IP) was detected by immunoblotting with anti-CEBPa.P65 was detected by immunoblotting with anti-P65.D. HEPG2 cells were treated with (+) or without (2) TNFa and co-immunostained with anti-CEBPa and anti-P65.E. Hepatocytes were transfected with a luciferase construct driven by the 23000 bp YKL40 promoter in addition to the control vector or vector expressing P65 or CEBPa or both.Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control.The numbers represent fold-change over the control empty vector (average of three independent experiments); error bars represent SD.Bottom panel, expression of P65 and CEBPa was verified by immunoblotting with anti-P65 and anti-CEBPa respectively.doi:10.1371/journal.pone.0050826.g004without TNFa and probed for P65 localization by immunostaining with anti-P65 (Figure 5B).In the untreated cells P65 remained exclusively cytoplasmic with little to no nuclear localization observed.In the TNFa treated cells, a significant amount of P65 translocated into the nucleus and quantification showed more than three fold nuclear abundance of P65 compared to the untreated cells (Figure 5C).Next, the HEPG2 cells were transfected with either scrambled siRNA or siRNA specific for human NOTCH1 and subjected to TNFa treatment.Immunostaining with anti-Notch1 showed more than four fold knockdown of NOTCH1 by siRNA (Figure S2B,C).Knockdown of NOTCH1 resulted in impairment of TNFa mediated nuclear translocation of P65 by two fold (Figures 5D andE).TNFa mediated nuclear localization of P65 in cells transfected with scrambled siRNA was not affected.This finding indicates that NOTCH1 acts as an upstream regulatory factor and controls TNFa mediated nuclear translocation of P65. ", "section_name": "NOTCH1 Regulates Nuclear Retention of NFKB/P65 in Response to TNFa", "section_num": null }, { "section_content": "Genomewide microarray analysis in our laboratory followed by miRNA gene expression analysis showed that miRNA-449a is downregulated more than two fold in HCV patients compared to non-HCV liver diseases and normals (Figure 1A).Computational target prediction of miRNA-449a using Targetscan (Targetscan.org)identified NOTCH1 to be a putative target for translational silencing.To test this, hepatocytes were transfected with either empty vector of vector expressing miRNA-449a and expression of both miRNA-449a and NOTCH1 were determined by qPCR.The results were normalized to GAPDH expression.Increased expression of miRNA-449a resulted in more than two fold downregulation of NOTCH1 (Figure 6A andB). Earlier we have shown that NFKB component P65, a protein regulated by NOTCH1, activates YKL40 expression through sequence specific promoter interaction (Figures 3 and4).It is likely that downregulation of miRNA-449a in HCV infected patients (Figure 1A) results in activation of NOTCH1/NFKB signaling that leads to upregulation of YKL40 expression.To test whether miRNA-449a regulates YKL40 expression, hepatocytes were transfected with either a control vector or vector expressing miRNA-449a along with an YKL40 promoter-driven luciferase reporter construct.In TNFa treated cells expression of YKL40 is reduced by more than two fold in the presence of miRNA-449a (Figure 6C, upper panel).qPCR analysis also showed downregulation of YKL40 by increased expression of miRNA-449a (Figure S3). Since, computational target analysis did not identify YKL40 to be a direct target for miRNA-449a; results obtained from promoter based reporter analysis suggest that miRNA-449a regulates the expression of YKL40 by silencing components of upstream transcriptional regulatory complexes such as NOTCH1/NFKB.To demonstrate that downregulation of YKL40 expression by miRNA-449a is mediated by silencing NOTCH1, hepatocytes were transfected with either scrambled siRNA or siRNA specific for NOTCH1 along with an YKL40driven luciferase reporter construct and cells were treated with TNFa.siRNA mediated knockdown of NOTCH1 impaired expression from the YKL40 promoter (Figure 6C, upper panel).We have demonstrated that nuclear P65, regulated by NOTCH1, activates YKL40 expression in response to TNFa.siRNA mediated knockdown of P65 also impaired expression from the YKL40 promoter (Figure 6C, upper panel).Western blot analysis using anti-YKL40 showed downregulation of YKL40 by expression of miRNA-449a or siRNA mediated knockdown of NOTCH1 or P65 in hepatocytes (Figure 6C, lower panels).qPCR analysis also confirmed downregulation of YKL40 by miRNA-449a (Figure S3).Immunoblot analysis using anti-NOTCH1 demonstrated that expression of miRNA-449a resulted in downregulation of NOTCH1.Since, P65 is a downstream factor for NOTCH1, knockdown of P65 did not affect its expression (Figure 6C, lower panels).This suggests that miRNA-449a regulates expression of YKL40 by modulating the NOTCH1 signaling pathway. To further determine if TNFa mediated activation of YKL40 is regulated by miRNA-449a, hepatocytes were transfected with either a control vector or vector expressing miRNA-449a along with an YKL40-driven luciferase reporter construct (23000 bp) and cells were treated with or without TNFa.In the cells expressing the empty vector, TNFa induced expression from the YKL40 promoter (Figure 6D).However, in presence of miRNA-449a this TNFa mediated upregulation of YKL40 was impaired. ", "section_name": "miRNA-449a Regulates YKL40 Expression by Modulating NOTCH1 Expression", "section_num": null }, { "section_content": "To determine whether downregulation of miRNA-449a in HCV infection is accompanied by upregulation of its target NOTCH1, biopsies were obtained from 10 chronic HCV patients, 10 alcoholic hepatitis patients, 10 NASH patients and 10 control normal donor livers at the time of liver transplant.Total RNA isolation followed by qPCR demonstrated NOTCH1 to be significantly upregulated in livers obtained from HCV patients.However, no significant difference in the expression of NOTCH1 was observed in alcoholic hepatitis patients, NASH patients and normal livers (Figure 7).Based on our in-vitro results obtained with hepatocytes, upregulation of NOTCH1 and YKL40 (Figure 1) in HCV patients can be attributed to downregulation of miRNA-449a. ", "section_name": "NOTCH1 Expression is Upregulated in HCV Patients", "section_num": null }, { "section_content": "YKL40, a member of the mammalian chitinase-like protein, has been shown to be elevated in patients with chronic liver diseases with fibrosis and cirrhosis (Figure 1) [5,6].In chronic liver disease patients, YKL40 expression has been shown to have a strong correlation with degree of fibrosis progression, extracellular matrix (ECM) synthesis, and serves an early indicator of liver fibrosis [23,24].In HCC patients, YKL40 expression is highly elevated in both serum and liver tissue [25].Here we demonstrate that YKL40 expression in human livers is regulated by co-operative action of several promoter-bound transcription factors.By computational analysis and subsequent in vitro studies we have defined putative binding sites for NFKB subunit P65 and CEBPa in the YKL40 promoter (Figure 3A).The NFKB pathway plays an important role in liver fibrosis, as its activation in hepatocytes can lead to activation of surrounding tissue macrophages and thus leading to fibrosis [26].We demonstrated that TNFa mediated YKL40 expression is regulated by P65, a component of the NOTCH/ NFKB signaling pathway (Figures 2 and3).Additionally our study demonstrates that NOTCH1 is essential for nuclear retention of P65 in human liver cells (Figure 5).Studies in animal models have shown that knockdown of NOTCH1 resulted in impairment of DNA binding and transcriptional activation ability of P65 and impacted dendritic cell differentiation [12].We have shown for the first time that the NFKB subunit P65 cooperates with CEBPa to regulate expression of YKL40 through direct DNA binding in hepatocytes (Figures 3 and4).Several studies have shown that CEBPa regulates activation of hepatic stellate cells which play key roles in hepatic fibrosis [27,28].Differential modulation of CEBPa has been shown in HCC patients [17,29].Our analysis on the regulation of the inflammatory biomarker YKL40 expression at the transcriptional level provides new insight into role of components of the NOTCH and NFKB signaling pathways in HCV induced hepatic fibrosis and HCC.It is of interest that HCV core protein NS3 can activate the NOTCH signaling pathway resulting in development of HCV-induced HCC [30].Activation of NOTCH signaling also promotes TGFb1 induced epithelial-mesenchymal transition, an initial step in the development of fibrosis, by directly interacting with the transcriptional machinery [31,32,33].Modulation of both NOTCH1 and NFKB pathways have also been implicated in several cancers including HCC [34,35,36,37]. In addition to demonstrating the interaction of NOTCH, CEBPa and NFKB pathways in YKL40 expression, we determined an important role for modulation of miRNA by HCV.The understanding of the complex role of miRNAs in the various physiological and pathological processes is still emerging.miRNAs have been implicated in regulation of pro-inflammatory cytokines, anti-inflammatory cytokines, and interferons [38].miRNA-21 has been shown to regulate chronic rejection and has been implicated in the development of fibrosis following liver transplantation [39,40].miRNA-21 modulates resident fibroblasts, epithelial cells and lymphocytes to produce pro-fibrotic cytokines resulting in deposition of ECM components [39,41].It has been shown that several liver specific miRNAs including miRNA-122, miRNA-148, miRNA-194 are sensitive biomarkers for hepatocyte injury and rejection after liver transplantation [42].In this study we identified a novel miRNA (miRNA-449a) that is modulated in HCV infection.Further, we have shown that miRNA-449a regulates HCV induced inflammatory responses (YKL40) implicated in allograft liver fibrosis. Previous studies in our laboratory have demonstrated an upregulation of autoimmune Th17 inflammatory cascade leading to liver fibrosis in HCV infection, particularly in recurrent HCV following orthotopic liver transplantation [43].Viral modulations of miRNA have been well known to influence transcriptional regulation in T cell responses, inflammation and fibrosis [44].Although suggested in literature, a direct effect of HCV mediated modulation of cellular inflammatory responses and fibrosis is yet to be determined.In our current study using promoter analyses techniques we provide direct evidence for the role of miRNA-449a, which is down regulated in HCV infection (Figure 1), in the upregulation pro-inflammatory YKL40 fibrotic cascade.miRNA-449a has been implicated transcriptional dysregulation affecting cell proliferation in several human diseases including cancers [45,46].In vitro studies have also shown that miRNA-449a can arrest cell proliferation and induce apoptosis [46,47].Thus, HCV induced down regulation of miRNA-449a in human livers can upregulate transcriptional factors leading to increased inflammatory response; promoting cell proliferation that can result in HCC. We have also demonstrated by in vitro analysis that miRNA-449a regulates TNFa mediated induction of YKL40 by targeting components of the NOTCH signaling pathway (NOTCH1) (Figure 6).We have shown for the first time in human hepatocytes that miRNA-449a targets NOTCH1 for translational silencing.Studies have shown that miRNA-34a is downregulated in patients with chronic hypoxia kidney diseases and promotes epithelialmesenchymal transition by targeting components of the NOTCH signaling pathway [48].In HCV infected patients expression of miRNA-449a was significantly downregulated (Figure 1A).In consistence with our in vitro results, in the same HCV patients downregulation of miRNA-449a was accompanied by significant upregulation of NOTCH1 (Figure 7).Thus, results obtained from patient samples and our in vitro analysis using hepatocytes indicate that upregulation of NOTCH1 resulting from downregulation of miRNA-449a stabilizes nuclear P65 to activate YKL40 expression in patients with HCV mediated hepatic fibrosis (Figure 1).The increased expression of YKL40 in patients with HCV mediated liver fibrosis [5,6] can be attributed to this novel pathway (Figure 8).Since, YKL40 is elevated in patients with multiple liver diseases (Figure 1B); it is likely that other parallel pathways for its transcriptional regulation may exist in non-HCV mediated liver fibrosis. Taken together our results provide new insight into the mechanisms by which miRNAs mediate changes in the inflammatory process by modulating components of the transcriptional machinery.The results from this study should assist in the development of novel strategies for identifying non-invasive biomarkers that can prognosticate patients and monitor those at increased risk for development of cirrhosis and HCC following HCV infection. ", "section_name": "Discussion", "section_num": null } ]	[ { "section_content": "The authors thank Ms. Billie Glasscock for her help in preparing this manuscript. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "This work was supported by the Barnes-Jewish Foundation at Barnes-Jewish Hospital, St. Louis, MO.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "", "section_name": "Supporting Information", "section_num": null } ]
10.1371/journal.pone.0200619	Proteomic analysis of canine oral tumor tissues using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC MS/MS) approaches	Les tumeurs buccales, y compris le mélanome buccal hautement invasif et métastatique (OM), le carcinome épidermoïde buccal non amygdalien (OSCC) et les tumeurs bénignes (BN), sont des néoplasmes courants chez les chiens. Bien que ces tumeurs se comportent différemment, des données limitées de leurs profils d'expression protéique ont été présentées, en particulier au niveau du protéome. La présente étude visait à i.) caractériser les empreintes peptidiques de masse (PMF) et identifier les candidats protéiques potentiels des sujets OM, OSCC, BN et témoins normaux, en utilisant la spectrométrie de masse à temps de vol par désorption/ionisation laser assistée par matrice (MALDI-TOF MS) et la spectrométrie de masse en tandem par chromatographie liquide (LC-MS/MS), ii.) identifier les candidats protéiques potentiels associés aux maladies, en utilisant la digestion en gel couplée à l'analyse spectrométrique de masse (GeLC-MS/MS) et iii.) rechercher les relations entre les médicaments de chimiothérapie et les protéines perturbées par la maladie. Un groupe distinct de chaque groupe d'échantillons et des PMF uniques avec des protéines candidates identifiées ont été révélés. Le fragment peptidique unique à 2 274 Da de la chaperone moléculaire de la sacsine (SACS) a été observé au stade précoce de l'OM, tandis que le fragment à 1 958 Da de la sous-unité alpha 10 du canal sodium voltage-dépendant (SCN10A) a été présenté au stade précoce et au stade avancé de l'OM. La masse peptidique à 2 316 Da de Notch1 est apparue dans les OM de stade précoce et les tumeurs buccales bénignes tandis que la masse peptidique à 2 505 Da de la sous-unité 3A de type N-méthyl-D-aspartate du récepteur ionotrope du glutamate (GRIN3A) a été identifiée dans tous les groupes. Les protéines marquées exprimées de GeLC-MS/MS comprenaient le domaine Jumonji contenant 1C (JMJD1C) dans les tumeurs bénignes, l'inversine (INVS) et le facteur 28 d'échange de nucléotides rho guanine (ARHGEF28) dans OM, le domaine BTB contenant 16 (BTBD16) dans OSCC, et la protéine tyrosine phosphatase non-récepteur de type 1 (PTPN1), BRCA2, la réparation de l'ADN associée (BRCA2), la protéine 2 de liaison au domaine WW (WBP2), le récepteur purinergique P2Y1 et la sous-unité 4 de l'activateur du protéasome (PSME4) dans tous les groupes cancéreux. Les connexions en réseau entre ces protéines et les médicaments de chimiothérapie, le cisplatine et la doxorubicine, ont également été démontrées. En conclusion, cette étude a dévoilé les FMP uniques et les nouveaux marqueurs protéiques candidats des tumeurs buccales canines.	[ { "section_content": "Oral neoplasms represent approximately 7% of all types of tumors in dogs [1].Among these, oral melanoma (OM) is the most aggressive, with high prevalence, accounting for 30-40% of all oral cancers [2,3] or 15-45% of all oral tumors [4].According to the World Health Organization (WHO) clinical staging scheme of OM, the prognosis is based on tumor size, lymph node involvement and distant metastasis or TNM system.Stage I is a tumor <2 cm in diameter; stage II is a 2 to <4 cm diameter tumor; stage III is a tumor !4 cm in diameter with or without lymph node metastasis, and stage IV is a tumor with distant metastasis [2].Several cases of OM were detected at the late stages (stages III and IV) with poor prognosis [5,6].OM cases generally recurred and/or metastasized rapidly after surgical resection [7].Oral squamous cell carcinoma (OSCC) is another common oral neoplasm in dogs, comprising 20-30% of all oral tumors [8].The frequently found non-tonsillar type is less aggressive than the tonsillar one [7].The histological grading of OSCC is defined as well, moderately and poorly differentiated [9].Well-differentiated SCC is similar to normal squamous epithelium, with compact laminated keratin or keratin pearls.For moderately and poorly differentiated SCC, greater degrees of mitotic activity and nuclear pleomorphism with less keratinization are present [9].For the benign tumors (BN), the non-malignant lesions are classified according to the origin of the cells, including peripheral odontogenic fibroma (fibromatous epulis of periodontal ligament origin), acanthomatous ameloblastoma (epithelial neoplasia of the enamel organ), odontoma (odontogenic neoplasm of mixed origin) and other odontogenic tumors [8].Several proteins have been reported to be potential biomarkers or therapeutic targets in canine oral tumors, such as melanoma cell adhesion molecule, cluster of differentiation 146 (CD146), Ras GTPase-activating-like protein IQGAP1, programmed death ligand-1, leptin, fascin-1, chondroitin sulfate proteoglycan-4 in OM, proliferating cell nuclear antigen, p63 and E-cadherin, high mobility group A2 in OSCC, and CD46 in ameloblastoma [10][11][12][13][14][15][16][17][18][19].However, our knowledge of protein expression involved in the development and progression of canine oral tumors is still limited, particularly in a large-scale analysis.Proteomics is the study of expressed proteins under a specific condition in large scale [20].Mass spectrometry (MS) is the high-throughput technology for protein profiling.A matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), composed of a MALDI source and a TOF mass analyzer, is used for searching peptide mass fingerprints (PMFs).MS spectra are obtained and compared to get fingerprints of ions that are characteristic of the cell/tissue/organism.In addition, three-dimensional principal component analysis (3D PCA) scatterplot has been used to reveal the uniformity and homogeneity of the sample group [21].MALDI-TOF MS was demonstrated as a rapid screening method to differentiate oral cancer, oral lichen planus, and chronic periodontitis in human saliva [22].In dog, MALDI-TOF was used to study different protein expression in tears from dogs with cancers (transmissible venereal tumor, mammary gland adenocarcinoma, squamous cells carcinoma, fibrosarcoma, etc.) and normal dogs in order to develop tear film analysis for cancer diagnosis and management in dogs [23].Specific mass spectra peaks on the PMF map can be further analyzed using MALDI-TOF/TOF MS, which was used to identify protein biomarkers in canine lymphoma, mammary tumor, prostate tumor and mast cell tumor [24][25][26][27][28][29][30][31].Another tandem MS, liquid chromatography-tandem mass spectrometry (LC-MS/MS), is used for routine identification of proteins.LC-MS/MS use electrospray ionization (ESI) whereas MALDI-TOF uses MALDI as an ionization source with different operation and performance characteristics [32].In-gel digestion coupled with mass spectrometric analysis (GeLC-MS/MS) is a onedimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by in-gel digestion and LC-MS/MS.GeLC-MS/MS is suitable for qualitative and quantitative complex protein identification [33].In dogs, this method was used to compare protein expression of formalin-fixed paraffin embedded and fresh-frozen sets of the same tissues [34].In addition, LC-MS/MS was used to measure plasma free metanephrine and free normetanephrine in dogs with pheochromocytoma for disease diagnosis and in lymphoma [30,35].There remain gaps in our knowledge of protein expression profiles of canine oral tumors, particularly at the proteome level. Since OM has high potential to metastasize, a combination of treatments is usually required for the late-stage OM, clinical stages III and IV.A number of chemotherapy drugs have been used to cure canine oral cancers after surgery.At the animal teaching hospital, Faculty of Veterinary Science, Chulalongkorn University, dogs with the late-stage OM undergo carboplatin chemotherapy with the median dosage 300 mg/m 2 for 6 times or else the metronomic therapy, continuous administration of low doses cyclophosphamide (10 mg/m 2 ) and standard dose piroxicam (0.3 mg/kg) as previously reported [36,37].Carboplatin, a derivative of the anti-cancer drug cisplatin, and doxorubicin (also called adriamycin) are common chemotherapy drugs used in canine oral cancer treatment, whereas cyclophosphamide and piroxicam have also been widely used in metronomic chemotherapy [36,38,39].In the present study, a fast and inexpensive bioinformatic tool was used to uncover functional relationships between drugs and disease proteins and fulfill experimental data.The proposed network maps demonstrated the molecular basis of disease, which could probably help select potential targets for early diagnosis, prognosis or effective treatment.The present study aimed to characterize PMFs of OM, OSCC, BN and normal control subjects, using MALDI-TOF and liquid chromatography tandem mass spectrometry (LC-MS/ MS), to identify potential protein candidates associated with the diseases, using GeLC-MS/ MS and to search for relationships between chemotherapy drugs and disease-perturbed proteins.Herein, we found a distinct cluster and a unique PMF in each canine oral tumor group.In addition, unique peptide fragment at 2,274 Da of sacsin molecular chaperone (SACS) was observed in early-stage OM, and at 1,958 Da of sodium voltage-gated channel alpha subunit 10 (SCN10A) in early-and late-stage OM.The peptide mass at 2,316 Da of Notch1 appeared in early-stage OM and benign oral tumors while the peptide mass at 2,505 Da of glutamate ionotropic receptor N-methyl-D-aspartate type subunit 3A (GRIN3A) was identified in all sample groups.We also found a peptide fragment at 3,039 Da of leucine-tRNA synthetase (LARS) in early-stage OM, OSCC, and benign oral tumors.Using GeLC-MS/MS, we discovered potentially novel candidate markers of canine oral tumors such as Jumonji domain containing 1C (JMJD1C or TRIP8) in benign tumors, inversin (INVS) and rho guanine nucleotide exchange factor 28 (ARHGEF28) in OM, BTB domaincontaining 16 (BTBD16) in OSCC, and protein tyrosine phosphatase non-receptor type 1 (PTPN1), BRCA2, WW domain binding protein 2 (WBP2), purinergic receptor P2Y1 variant 2 (P2Y1) and proteasome activator subunit 4 (PSME4) in all cancerous groups.We also demonstrated relationships between cisplatin and doxorubicin and disease-perturbed proteins, whereas cyclophosphamide/piroxicam showed no or very faint relationships with most proteins.These data might help veterinarians choose drugs of choice and treatment plan. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Materials and methods", "section_num": null }, { "section_content": "Tumor tissue samples were collected from patients undergoing surgery at the Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University.They were comprised of 15 OM (7 early-and 8 late-stage OM), 7 OSCC and 8 benign oral tumors (age range 1-16 years).Eight normal gingiva tissue samples were collected from fresh dog carcasses with no history or clinical signs of oral cavity or cancerous problems (age range 8-9 years).OM was classified according to the TNM staging system of the WHO [2].Stages I and II were defined as an early-stage OM, whereas stages III and IV were determined as the late-stage OM [40].The sample collection protocol was approved by the Chulalongkorn University Animal Care and Use Committee (CU-ACUC) and samples were obtained with the consents of owners.Samples were bisected: one half was fixed in 10% neutral buffered formalin for histopathological diagnosis; and the other half was stored in RNAlater (Thermo Fisher Scientific, Waltham, MA) at -20˚C for proteomic analysis, for which approximately 100 mg of tissues were pulverized in liquid nitrogen and incubated in 0.5% SDS for 1 h at room temperature, followed by centrifuging at 12,000 rpm for 15 min.Supernatants were kept at -20 ˚C until further analysis. ", "section_name": "Sample collection", "section_num": null }, { "section_content": "Formalin-fixed, paraffin-embedded (FFPE) sections of 4-μm thickness were stained with hematoxylin and eosin (H&E) for routine histopathological diagnosis.To confirm amelanotic melanoma, tissues were deparaffinized with xylene and rehydrated through a series of graded concentrations of ethanol in water.The sections were antigen retrieved in 0.01 M sodium citrate, pH 6.0, in a microwave oven (800 W) for 10 min.Endogenous peroxidase activity was quenched by incubating the slides in H 2 O 2 3% in methanol at room temperature for 10-20 min.Non-specific immunoglobulin binding was blocked with 1-3% (w/v) bovine serum albumin (Merck, Rockland, MA) at 37˚C for 20 min.Sections were incubated with 1:50 mouse monoclonal against human Melan-A antibody (Dako, Glostrup, Denmark M7196), at 4 ˚C for 16 h.A polymer-based non-avidin-biotin system, the EnVision detection system system (Dako), was used for detection of the reaction, and labeling was visualized with a 3,3 0 -diaminobenzidine tetrahydrochloride (DAB) substrate kit (Dako).The sections were counterstained with Mayer's hematoxylin.Canine melanotic melanoma was used as a positive control.The positive areas were seen in cytoplasmic areas. ", "section_name": "Histopathology and immunohistochemistry", "section_num": null }, { "section_content": "Total protein concentrations were determined by Lowry's assay at 690 nm, using bovine serum albumin as a standard [41].Protein samples in each group were pooled, dried and redissolved in 100% acetonitrile (ACN) containing 5% trifluoroacetic acid (TFA).The samples of 1 μg/μL were mixed with MALDI matrix solution [10 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) in 100% ACN containing 5% TFA] at the ratio of 1:1, spotted as 20 individual replicates on to a MTP 384 ground steel target plate (Bruker Daltonics, Billerica, MA) and air dried.Mass spectra were acquired on the Ultraflex III TOF/TOF (Bruker Daltonics) in a linear positive mode over a mass range 1,000-20,000 Da.The standard peptide mixtures of Proteo-Mass Peptide & Protein MALDI-MS Calibration Kit (Sigma Aldrich, St. Louis, MO) were used for the external calibration, including human angiotensin II (m/z 1,046), P14R (m/z 1,533), human adrenocorticotropic hormone fragment 18-39 (m/z 2465), bovine insulin oxidized B chain (m/z 3,465), bovine insulin (m/z 5,731), and cytochrome c (m/z 12,362).Each spectrum was obtained from 500 laser shots, with a 50 Hz laser.Fingerprint spectra, pseudo-gel view and 3D PCA scatterplot were analyzed by ClinProTools version 3.0 and flexAnalysis version 3.3 software (Bruker Daltonics), respectively [22,42].To analyze candidate mass spectra between 1,000 and 20,000 Da, three statistical algorithms incorporated in the ClinProTools software version 3.0, including Quick Classifier (QC)/ Different Average, Supervised Neural Network (SNN) and the Genetic Algorithm (GA), were utilized.The recognition capability and cross validation values >90% were used to reveal the reliability of the candidate peak selection [43].And in order to analyse intensity values, three statistical tests (Anderson-Darling (AD), t-test/ ANOVA (TTA), and Wilcoxon/Kruskal-Wallis (W/KW), incorporated into ClinProTools software version 3.0, were used.Results with p<0.05 were considered significant.To analyze specific peptide sequences, peptide samples were purified using C18 ZipTip (Merck Millipore, Darmstadt, Germany) and eluted with 2% series of ACN.After that samples were analyzed by LC-MS/MS using an Ultimate 3000 LC System (Thermo Scientific Dionex, Waltham, MA, US) on a nanocolumn PepSwift monolithic column 100 mm i.d.650 mm, at a flow rate of 300 nL/min.The nanoLC system was connected to an electrospray interface with ESI-Ion Trap MS (Bruker Daltonics).The LC-MS raw data were converted into an mz XML file by CompassXport software (Bruker Daltonics).All data were obtained for quantification based on MS signal intensities of individual analysis using DeCyder MS differential analysis software (DeCyder MS, GE Healthcare, Amersham, UK).All MS/MS data from DeCyder MS software were submitted to a database search against the NCBI Canis lupus familiaris database using MASCOT software version 2.2 (Matrix Science, London, UK). ", "section_name": "Analysis of peptide patterns by MALDI-TOF MS", "section_num": null }, { "section_content": "For the protein identification by GeLC-MS/MS, 50 μg of protein pools were fractionated on 12% SDS-PAGE (Atto, Tokyo, Japan).After Coomassie Brilliant Blue R-250 (CBB R-250) protein staining and destaining with 16.5% ethanol in 5% acetic acid, the gel was scanned using a GS-710 scanner (Bio-Rad, Benicia, CA) and stored in 0.1% acetic acid until in-gel tryptic digestion, where protein bands were divided into 25 segments per lane according to size and chopped into 1 mm 3 pieces.Gel plugs were dehydrated using 100% ACN for 5 min and dried for 15 min at room temperature.Disulfide bonds were reduced by 10 mM dithiothreitol (DTT) in 10 mM ammonium bicarbonate for 1 h at room temperature and alkylated in 100 mM iodoacetamide (IAA) in 10 mM ammonium bicarbonate for 1 h at room temperature in the dark.The gel pieces were dehydrated twice in 100% ACN for 5 min and trypsin-digested overnight at 37 ˚C.The sequencing grade modified trypsin (Promega, Madison, WI) was used.The tryptic digestion was performed in 50mM NH 4 HCO 3 (pH 7.8) as recommended by the manufacturer.The tryptic peptides were extracted from the gels using 50% ACN in 0.1% formic acid (FA).Finally, peptide mixtures were dried and kept at -80 ˚C until LC-MS/MS analysis. Prior to sample injection into LC-MS/MS, dried extracted peptides were dissolved in 0.1% FA in LC/MS-grade water and centrifuged at 12,000 × g for 10 min.The peptide solutions were analyzed by an Ultimate 3000 LC System (Thermo Scientific Dionex, Sunnyvale, CA).PepSwift monolithic nanocolumn 100 mm i.d.650 mm at a flow rate of 300 nL/min, using a multi-step gradient of a 10-90% linear concentration of 80% ACN in 0.1% FA within 20 min.The nanoLC system was connected with an electrospray interface with ESI-Ion Trap MS (Bruker Daltonics).The LC-MS raw data were converted into an mz XML file by CompassXport software (Bruker Daltonics).All data were obtained for quantification based on MS signal intensities of individual analysis using DeCyder MS differential analysis software (DeCyder MS, GE Healthcare, Amersham, UK).ANOVA statistical analysis, incorporated into the DeCyder MS, was used to identify significantly varying peptides among different sample groups. All MS/MS data from DeCyder MS software were submitted to a database search against the NCBI Canis lupus familiaris database using MASCOT software version 2.2 (Matrix Science, London, UK).Proteins were classified according to their molecular function, biological process and cellular component using PANTHER classification system version 8.1 [44].Protein list comparison among different sample groups was displayed using jvenn diagram [45].Proteins that were individually expressed in each group were chosen as candidate proteins.Then, the list of candidate proteins was analyzed for their relationship with carcinogenesis and chemotherapy drugs by the online-based software Stitch version 5.0 [46].Hierarchical clustering heat map was performed using Multiexperiment Viewer (MeV) program, version 4.8, with the Pearson correlation distance metric [47].The statistical significance level was set at p<0.05. ", "section_name": "Protein identification by GeLC-MS/MS", "section_num": null }, { "section_content": "The histopathological classifications of the OM and samples are shown in Table 1 and S1 Fig. For the OSCC, 2 and 5 samples were diagnosed as poorly differentiated and well differentiated, respectively.The benign oral tumors comprised 4 acanthomatous ameloblastomas and 4 peripheral odontogenic fibromas. Different PMFs of normal gingiva tissues, early-stage OM, late-stage OM, OSCC and benign tumors were detected in the range 1,000-10,500 Da (Fig 1).A number of unique peaks distinguishing each group were observed in addition to some common peaks among different groups as demonstrated in Table 2.The 3D view of the plot analysis of PCA scores exhibited a discrete cluster of each sample group that was clearly distinguished from the others, indicating similarity within a group (Fig 2).The MALDI-TOF MS results had an accurate outcome with the 95% confidence interval.The cross validation, calculated by Kruskall-Wallis test, in the normal controls, early-stage OM, late-stage OM, OSCC and benign oral tumors was 100%, 100%, 94.87%, 100% and 100%, respectively, and the recognition capability, calculated by Kruskall-Wallis test, in the normal controls, early-stage OM, late-stage OM, OSCC and benign oral tumors was all 100%, indicating that the results were of high reliability. Proteins expressed high signal intensities either individually found in each sample group or commonly found in normal and benign tumor, early-and late-stage OM, and only cancerous groups were analyzed by LC-MS/MS.In addition, protein lists were submitted to the PAN-THER classification system.The results showed the association of these proteins with the molecular function, biological process and cellular component (Table 3).Proteins function through interaction with other proteins or molecules.Networks of protein-protein and protein-chemotherapy drug interactions were analyzed by the Stitch program, version 5.0.Edge confidence scores were used to represent the strength of the protein-protein interactions at the functional level.Pathways with high edge confidence scores (>0.700) were demonstrated In addition, the PANTHER classification system was used to reveal the association of these proteins with the molecular function, biological process and cellular component (Table 4 and S1 Table ).In the S1 Table, the relative expression levels of distinct proteins in normal controls, benign tumors, early-and late-stage oral melanoma (OM) and oral squamous cell carcinoma (OSCC) were shown as log2 intensities [48].Protein scores (ID scores) were derived from ion scores as a non-probabilistic ranking protein hits and obtained as the sum of peptide 9).Except ARHGEF28, no or very faint correlation of target proteins with cyclophosphamide/piroxicam, common drugs used in metronomic therapy, was observed.And no correlation of all drugs and BTBD16 was exhibited as well as the correlation of the drugs and a number of proteins in an all cancer group, including, teneurin transmembrane protein 4 (TENM4), coiled-coil domain containing 191 (KIAA1407), NK6 homeobox 1 (NKX6-1), ral GTPase-activating protein subunit alpha-1 (RALGAPA1), semaphorin 3C (SEMA3C), chloride voltage-gated channel 4 (CLCN4) and family with sequence similarity 92 member B (FAM92B).However, the reason may be due to the fact that the connection of those proteins and chemotherapy drugs has never been discovered. ", "section_name": "Results", "section_num": null }, { "section_content": "This study initially used the top-down MS-based approach, MALDI-TOF MS, to exhibit PCA plots and PMFs of OM, OSCC, BN and normal control groups.Identification of proteins underlying discriminatory peaks was performed by LC-MS/MS.The bottom-up GeLC-MS/ MS approach was used to identify protein markers in each group.Different PMFs and a number of unique peaks as well as a discrete cluster from the PCA analysis were exhibited in each sample group.Hence, MALDI-TOF MS can possibly be used as rapid and reliable diagnostic tools for detection of canine oral tumors.MALDI analysis has been used for the diagnosis of human head and neck squamous cell carcinoma and human OSCC from oral brush biopsy and oral fluid [49][50][51].Since we could distinguish early-and late-stage OM by MAL-DI-TOF MS, the clinical application of this technique in early detection of the disease, which implies the early treatment, the better quality of life of the patients and probably higher survival rate, would be possible.The technique is suitable to fulfill the diagnosis since the conventional histological analysis had lengthy processes and required experts in veterinary pathology to interpret results where discordance usually exists among specialists.In fact, histologydirected analysis of tissue sections which combines tissue section and MALDI-TOF MS to target specific cells are currently interested [52].However, more number of clinical samples is required to set databanks of PMFs and PCA plots. This study also revealed the candidate proteins from MALDI mass spectra.SACS is a cochaperone of heat shock protein (HSP)70.It is required for proper folding and function of HSP70 chaperone proteins [53].HSP70 was target therapy for cancers since a number of cancers overexpress HSP70 family members [54,55].However, the association of SACS and HSP in canine oral cancer has not been reported.SCN10A, exclusively identified in OM groups, is one of the voltage-gated sodium channels (VGSC) proteins.The alteration of the Na + ions at the cell membrane is important for cell proliferation, especially cancer cells [56].Enhanced protein expression in a VGSC group, such as SCN5A in human breast cancer and SCN4A in prostate cancer, was reported to be associated with cancer invasiveness [57,58].Thus, the SCN10A might affect the canine OM progression.Predicted interaction between SACS or SCN10A proteins and chemotherapy drugs, cyclophosphamide/piroxicam, was exhibited in the present study via several cytochrome P450 (CYP) proteins which plays an important role in cancer development and response to therapy [59][60][61][62]. For Notch1, the protein in the Notch signaling pathway, it was also involved in tumor progression [63].Notch1 associated with epithelial-mesenchymal transition (EMT) in prostate cancer.Inhibition of Notch1 decreased the proliferation of melanoma cell line [64,65].In the present study, Notch 1 expression was found in the early-stage OM and benign oral tumors, the relationship of Notch 1 expression and early detection of OM should be further investigated.LARS, another candidate found in several tumor groups, was previously discovered as the anticancer target due to the inhibition of nutritional pathway of cancer cells [66].Hence, the inhibition of LARS in oral cancers and tumors in dogs should be further investigated for the possibility to be developed as targeted therapy.However, all of the candidate biomarkers from this study should be confirmed by other techniques such as western blotting. With GeLC-MS/MS, a number of proteins were shown to be increasingly expressed in canine oral tumors whereas GAPDH was remarkably observed only in normal controls, indicating it might not be a suitable housekeeping gene/protein for gene expression study in oral cavity in dogs.As GAPDH functions to generate NADH from NAD + in glycolysis pathway.In general, increased GAPDH expression was shown in cancer owing to the NAD + supply by lactate dehydrogenase in anaerobic glycolysis [67].However, in our case, lack of GAPDH in tumor cells may be due to depleted NAD + , probably indicating aberrant anaerobic glycolysis in canine oral tumors.JMJD1C isoform X5 was found to be increased in the benign tumors.JMJD1C plays an important role in the histone demethylation as an epigenetic regulation.Decreased expression of the JMJD1C variant, s-JMJD1C, was observed in breast cancer whereas in normal breast tissues the expression was significantly increased, suggesting its functions in tumor suppression [68].In our study, JMJD1C was solely found in benign tumors, not in any cancers or normal tissues, probably indicating a potential role of JMJD1C as a biomarker for benign tumors of the oral cavity in dogs. In both early-and late-stage OM, INVS isoform X6 was found to be overexpressed.As INVS has two IQ calmodulin (CALM) domains, we added keywords: inversin, calmodulin 1, calmodulin 2, calmodulin 3 and cisplatin or doxorubicin or cyclophosphamide/piroxicam, to the Stitch program [69].A link between INVS, a unique protein in an OM group, and cisplatin or doxorubicin via CALM2, calcium ions, and tumor protein p53 (TP53) or nitric oxide synthase 3 (NOS3) was proposed.CALM is the calcium-binding protein that regulates cellular proliferation.As CALM is a multi-phase protein, paradoxical roles of CALM have been reported.CALM inhibition could induce proliferative arrest and apoptosis mediated by activation of a tumor suppressor, TP53, and restore resistant cell sensitivity to chemotherapy drugs such as doxorubicin [70,71], whereas CALM and calcium played an important role in cisplatin-induced tumoricidal activity of peritoneal macrophages in mouse [72].The low levels of NOS, a CALM -dependent enzyme, also participated in the induction of cell proliferation, as NOS and CALM inhibitors could inhibit this process [70].In OSCC, BTBD16 was markedly expressed.The BTB/POZ domain is a common structural domain in several proteins.BTBD7 enhanced hepatocellular carcinoma (HCC) angiogenesis and metastasis, hence, promoting HCC progression [73].Zinc finger and BTB domain-containing protein 3 played an important role in the in human melanoma, lung carcinoma, and breast carcinoma cell growth via the reactive oxygen species (ROS) detoxification pathway [74].The rho guanine nucleotide exchange factor 28 (ArhGEF28 or p190RhoGEF or Rgnef), a member of the Dbl family of Rho-GEFs which promote the active GTP-bound state of Rho GTPases, was found to be expressed in early-and late-stage OM [75,76].Elevated ArhGEF28 expression promotes colorectal carcinoma invasion and tumor progression via interaction with focal adhesion kinase [77,78].Arh-GEF28 interacts with RHOA which was found to be overexpressed in several cancers such as prostate cancer [79], gastric cancer [80] and chronic myeloid leukemia [81].Decreased RhoA protein expression by RhoA small interfering RNA was associated with the increased sensitivity to doxorubicin in human colon cancer cell line [82]. A number of expressed proteins in the cancerous group (OM in all stages and OSCC) were found in protein-chemotherapy drug interactions (cisplatin and doxorubicin) including PTPN1, BRCA2, WBP2, P2Y1 and PSME4 (Figs 789).PTPN1 and BRCA2 expression was possibly a negative feedback of the tumorigenesis.PTPN13 inhibited hepatocellular carcinoma through inactivation of the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK) signaling pathway [83].The elevated expression of epidermal growth factor (EGF), EGFR, Janus kinase (JAK) and proto-oncogene tyrosine-protein kinase Src (Src) is noted in human oral cancer [84].Activation of EGFR/Erk1/2 and JAK could enhance invasiveness of cisplatin-resistant ovarian cancer cells in vitro, and the inhibition of both EGFR and JAK appeared to be an efficient approach to treat human ovarian cancer [85,86].Increased EGFR expression was related to doxorubicin resistance in lung cancer cells [87].BRCA2 is a tumor suppressor that prevent cells from growing and dividing too rapidly [88].Mutation of BRCA2 in breast and ovarian cancers compromises DNA homologous repair and then leads to BRCA-associated tumors sensitive to cisplatin, which causes DNA breaks and requires a repair process [89].However, secondary mutation of BRCA2 can elicit cisplatin resistance in ovarian carcinomas [90]. WBP2 is a tyrosine kinase substrate.The enzyme can phosphorylates targets and induce tumorigenesis [91].In human breast cancer, phosphorylation of WBP2 at Tyr192 and Tyr231 was regulated by c-Src and c-Yes kinases and was stimulated by EGF.WBP2 tyrosine phosphorylation could enhance the transcription of estrogen receptor α, which induced the angiogenesis of breast cancer [92,93].The P2Y1 receptor belongs to a family of G protein-coupled receptor.In human prostate cancer and melanoma, the P2Y1 receptor induces cell apoptosis and/or inhibits cell proliferation and is a putative target for cancer therapy [94,95].The role of P2Y1 receptor in canine oral cancer requires further investigation.Proteasomes, a multisubunit protein complex, function to destroyed unnecessary or damaged proteins.Enriched proteasome genes, including PSME4 and PSMD14, was exhibited in doxorubicin-derived resistant ovarian cancer cell line, suggesting that this proteasome pathway may be involved in the development of resistance to doxorubicin [96].As various drugs have been used to treat dogs with oral malignant tumors following surgical excision, in the present study we demonstrated relationships between chemotherapy drugs and disease-perturbed proteins, which might help veterinarians choose drugs of choice and treatment plan.Further studies are required for the effects of drugs on the protein biomarkers of the diseases. In our study, we obtained different expressed proteins from MALDI-TOF MS combined with LC-MS/MS and from GeLC-MS/MS.The plausible explanations included different types of ionization techniques, the sample preparation steps and the statistical analysis.The reason why proteins from GeLC MS/MS did not appear in results from MALDI-TOF MS combined with LC-MS/MS might be associated with the ZipTip cleanup in the sample preparation steps for MALDI-TOF MS.Since peptide masses were ZipTip purified and eluted through the 2% series of ACN, the amount of each peptide mass was lessen and might not be able to be detected by LC-MS/MS technique whereas the GeLC-MS/MS could identify proteins from intact peptides.On the other hand, the reason why proteins from MALDI-TOF MS combined with LC-MS/MS did not appear in results from GeLC MS/MS was probably due to T-test/ ANOVA statistics (p<0.05)performed to select significant proteins in data processing of GeLC-MS/MS.Therefore, the candidate proteins from MALDI-TOF MS combined with LC-MS/MS might not be present in GeLC-MS/MS analysis.A limitation of the current study was probably the inability to specify whether the peptides were from the tumor cells, stroma or elsewhere (e.g., interstitial fluid) because in our study we did not perform primary culture or using the flow cytometer to separate solely tumor cells for protein extraction. ", "section_name": "Discussion", "section_num": null }, { "section_content": "The present study revealed the distinct cluster of each sample group, unique PMFs and protein identification in OM, OSCC, BN and normal control subjects, using MALDI-TOF MS combined with LC-MS/MS, and also identified potential protein candidates associated with the diseases, using GeLC-MS/MS.The network connections between these proteins and the chemotherapy drugs, cisplatin, doxorubicin, cyclophosphamide and piroxicam, were also demonstrated.For future work, protein-protein interaction in the diseases should be confirmed by high-throughput approaches, such as yeast two-hybrid screening and affinity purification coupled to mass spectrometry [97,98]. ", "section_name": "Conclusions", "section_num": null } ]	[ { "section_content": "We sincerely thank Assoc.Prof. Dr. Anudep Rungsipipat for histopathology advice and Dr. Sekkarin Ploypetch for sample collection.Special thanks to Miss Nathamon Yimpring, Miss Atchara Paemanee and Miss Suthathip Kittisenachai for technical assistance. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "This work was supported by: Chase Enterprise (Siam) Co., Ltd., grant number RES_60_229_31_025 (http://www.chasesiam.com/th) (GS -funding receiver); Faculty of Veterinary Science, Chulalongkorn University, grant number RG 1/2559 (http://www.vet.chula.ac.th/ vet2014/) (GS -funding receiver); The 100th doxorubicin, were also demonstrated.In conclusion, this study unveiled the unique PMFs and novel candidate protein markers of canine oral tumors. Anniversary Chulalongkorn University for Doctoral Scholarship (http://www.grad.chula.ac.th/th/01_ information/c01_scholarship_detail_100ys_eng.php) (SP -funding receiver); The 90th Anniversary of Chulalongkorn University Scholarship (http:// www.grad.chula.ac.th/th/01_information/c01_ scholarship_detail_90yearfund_eng.php)(SPfunding receiver); Overseas Research Experience Scholarship for Graduate Student, (http://www.grad.chula.ac.th/th/01_information/c01_ scholarship_detail_OverseasResearch_eng.php)(SP -funding receiver).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "All relevant data are within the paper and its Supporting Information files. ", "section_name": "", "section_num": "" }, { "section_content": "Conceptualization: Gunnaporn Suriyaphol. Formal analysis: Gunnaporn Suriyaphol. Funding acquisition: Sirinun Pisamai, Gunnaporn Suriyaphol. Methodology: Sirinun Pisamai, Sittiruk Roytrakul, Narumon Phaonakrop, Janthima Jaresitthikunchai. Project administration: Gunnaporn Suriyaphol. Resources: Sirinun Pisamai. Software: Sittiruk Roytrakul, Narumon Phaonakrop, Janthima Jaresitthikunchai. Supervision: Sittiruk Roytrakul, Gunnaporn Suriyaphol. Validation: Sirinun Pisamai, Sittiruk Roytrakul, Gunnaporn Suriyaphol. Visualization: Sirinun Pisamai. Writing -original draft: Gunnaporn Suriyaphol. Writing -review & editing: Sirinun Pisamai, Gunnaporn Suriyaphol. ", "section_name": "Author Contributions", "section_num": null }, { "section_content": "Conceptualization: Gunnaporn Suriyaphol. ", "section_name": "Author Contributions", "section_num": null }, { "section_content": "Formal analysis: Gunnaporn Suriyaphol. Funding acquisition: Sirinun Pisamai, Gunnaporn Suriyaphol. ", "section_name": "Data curation: Sirinun Pisamai.", "section_num": null }, { "section_content": "Methodology: Sirinun Pisamai, Sittiruk Roytrakul, Narumon Phaonakrop, Janthima Jaresitthikunchai. Project administration: Gunnaporn Suriyaphol. Resources: Sirinun Pisamai. Software: Sittiruk Roytrakul, Narumon Phaonakrop, Janthima Jaresitthikunchai. Supervision: Sittiruk Roytrakul, Gunnaporn Suriyaphol. Validation: Sirinun Pisamai, Sittiruk Roytrakul, Gunnaporn Suriyaphol. Visualization: Sirinun Pisamai. Writing -original draft: Gunnaporn Suriyaphol. Writing -review & editing: Sirinun Pisamai, Gunnaporn Suriyaphol. ", "section_name": "Investigation: Sirinun Pisamai.", "section_num": null } ]
10.1371/journal.pone.0238262	Association between tumor mutation profile and clinical outcomes among Hispanic Latina women with triple-negative breast cancer	Triple-negative breast cancer (TNBC) represents 15%-20% of all breast cancer types. It is more common among African American (AA) and Hispanic-Latina (HL) women. The biology of TNBC in HL women has been poorly characterized, but some data suggest that the molecular drivers of breast cancer might differ. There are no clinical tools to aid medical oncologists with decisions regarding appropriate individualized therapy, and no way to predict long-term outcomes. The aim of this study was to characterize individual patient gene mutation profiles and to identify the relationship with clinical outcomes. We collected formalin-fixed paraffin-embedded tumors (FFPE) from women with TNBC. We analyzed the gene mutation profiles of the collected tumors and compared the results with individual patient's clinical histories and outcomes. Of 25 patients with TNBC, 24 (96%) identified as HL. Twenty-one (84%) had stage III-IV disease. The most commonly mutated genes were TP53, NOTCH1, NOTCH2, NOTCH3, AKT, MEP3K, PIK3CA, and EGFR. Compared with other international cancer databases, our study demonstrated statistically significant higher frequencies of these genes among HL women. Additionally, a worse clinical course was observed among patients whose tumors had mutations in NOTCH genes and PIK3CA. This study is the first to identify the most common genetic alterations among HL women with TNBC. Our data strongly support the notion that molecular drivers of breast cancer could differ in HL women compared with other ethnic backgrounds. Therefore, a deeper understanding of the biological mechanisms behind NOTCH gene and PIK3CA mutations may lead to a new treatment approach.	[ { "section_content": "FoundationOne®CDx (F1CDx) is a next generation sequencing based in vitro diagnostic device for detection of substitutions, insertion and deletion alterations (indels), and copy number alterations (CNAs) in 324 genes and select gene rearrangements, as well as genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens.The test is intended as a companion diagnostic to identify patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling.Additionally, F1CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms.The F1CDx assay is a single-site assay performed at Foundation Medicine, Inc. ", "section_name": "Intended Use", "section_num": null }, { "section_content": "", "section_name": "Table 1: Companion diagnostic indications", "section_num": null }, { "section_content": "The test is also used for detection of genomic loss of heterozygosity (LOH) from formalin-fixed, paraffin-embedded (FFPE) ovarian tumor tissue.Positive homologous recombination deficiency (HRD) status (defined as tBRCA-positive and/or LOH high) in ovarian cancer patients is associated with improved progression-free survival (PFS) from Rubraca (rucaparib) maintenance therapy in accordance with the RUBRACA product label. ", "section_name": "Technical Specifications", "section_num": null }, { "section_content": "Genes with full coding exonic regions included in FoundationOne®CDx for the detection of substitutions, insertion-deletions (indels), and copy-number alterations (CNAs).FoundationOne CDx is a next-generation sequencing based in vitro diagnostic device for detection of substitutions, insertion and deletion alterations, and copy number alterations in 324 genes and select gene rearrangements, as well as genomic signatures including microsatellite instability (MSI) and tumor mutational burden (TMB) using DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens.For the complete intended use statement, including companion diagnostic indications and warnings and limitations, please see the FoundationOne CDx Technical Information, www.foundationmedicine.com/f1cdx. ", "section_name": "Current Gene List²", "section_num": null } ]	[]
10.18632/oncotarget.7000	Splicing factor mutations predict poor prognosis in patients with <i>de novo</i> acute myeloid leukemia	Mutations in splicing factor (SF) genes are frequently detected in myelodysplastic syndrome, but the prognostic relevance of these genes mutations in acute myeloid leukemia (AML) remains unclear. In this study, we investigated mutations of three SF genes, SF3B1, U2AF1 and SRSF2, by Sanger sequencing in 500 patients with de novo AML and analysed their clinical relevance. SF mutations were identified in 10.8% of total cohort and 13.2% of those with intermediate-risk cytogenetics. SF mutations were closely associated with RUNX1, ASXL1, IDH2 and TET2 mutations. SF-mutated AML patients had a significantly lower complete remission rate and shorter disease-free survival (DFS) and overall survival (OS) than those without the mutation. Multivariate analysis demonstrated that SFmutation was an independent poor prognostic factor for DFS and OS. A scoring system incorporating SF mutation and ten other prognostic factors was proved very useful to risk-stratify AML patients. Sequential study of paired samples showed that SF mutations were stable during AML evolution. In conclusion, SF mutations are associated with distinct clinic-biological features and poor prognosis in de novo AML patients and are rather stable during disease progression. These mutations may be potential targets for novel treatment and biomarkers for disease monitoring in AML.	[ { "section_content": "mutations and small cohorts studied, the association of SF mutations with clinic-biologic features and their prognostic implication in de novo AML patients remain unclear.Further, there has been no report in literature concerning the stability of SF mutations in AML. In this study, we assessed the clinical implication of SF mutations in 500 unselected adults with de novo AML and their interactions with other 18 genetic alterations.Longitudinal follow-ups of the status of SF mutations during the clinical course were also performed in 163 patients to investigate the stability and pathogenic role of these mutations in AML.To the best of our knowledge, this is the first study to address the prognostic implication of SF mutations in a large cohort of patients with de novo AML.We found that SF mutation was an independent poor-risk factor for overall survival (OS) and disease-free survival (DFS) in these patients. ", "section_name": "", "section_num": "" }, { "section_content": "Mutations of the RNA splicing machinery genes were identified in 54 (10.8%) of 500 patients, including 12 (2.4%) with SF3B1 mutations, 15 (3.0%) with U2AF1 mutations, and 27 (5.4%)with SRSF2 mutations, respectively (Table 1).SF mutations in all these patients were heterozygous.None had two of these three SF mutations at the same time, suggesting these three mutations were mutually exclusive (Table 1, Figure 1 and Supplementary Table 4). The most common SF3B1 mutation was K666M (n = 5), followed by K700E (n = 3) (Table 1).Ring sideroblasts could be detected in two (33%) of the six patients who had bone marrow smears for iron staining.Regarding U2AF1 mutations, ten patients had exon 2 mutations, including S34F in six patients, S34Y in three and S34T in one; six patients had exon 5 mutations, including Q157P in three patients, Q157R in two and E159_M160insYE in one.One patient (patient 18) had concurrent exon 2 S34F and exon 6 Q157R mutations.Among the 27 SRSF2-mutated patients, 24 patients had missense mutations, including P95H in 12 patients, P95L in 8 and P95R in 4. Two patients (patients 28 and 54) had P95_R102del (c.284_307del), a 24-base pair deletion, and Abbreviations: UPN, unique patient number; FAB, French-American-British; U, undetermined.the remaining one patient (patient 48) had R94_p95insR (c.283_284insGCC), a 3-base pair insertion (Table 1) ", "section_name": "results sF mutations in patients with de novo AMl", "section_num": null }, { "section_content": "SF-mutated patients were older (median, 67.5 years vs. 49 years, P < 0.0001, Table 2), male predominant (14.4% in males vs. 6% in females, P = 0.0033) and had a lower incidence of FAB M2 subtype (P = 0.0499) than other patients.The SF mutations were positively associated with the expression of HLA-DR (P = 0.0156) and CD34 (P = 0.0131), but inversely associated with the expression of CD33 (P = 0.0379) and CD56 (P = 0.0493) on the leukemic cells (Supplementary Table 1).Correlation of the clinical and laboratory features with mutations in individual SF genes was shown in Supplementary Table 2. ", "section_name": "correlation of sF mutations with clinical and laboratory features", "section_num": null }, { "section_content": "Chromosome data were available in 482 patients at diagnosis, including 51 SF-mutated and 431 SFwild patients (Supplementary Table 3).SF mutations occurred more frequently in patients with intermediaterisk cytogenetics (13.2%) than in those with favorable-or unfavorable-risk cytogenetics (5.5%, P < 0.0001).None of the patients with t(8;21), inv (16), or t(7;11) showed SF mutation, but one patient with t(15;17) harbored this mutation concurrently.There was no association of SF mutations as a whole with other chromosomal abnormalities, including +8, +11, +13, +21, -5/del(5q), and -7/del(7q).Intriguingly, U2AF1 mutations occurred frequently in patients with -7/7q-(P = 0.0352) A score of -3 was assigned for NPM1 + /FLT3-ITD -and -2 for CEBPA double-mutation and IDH2 mutation whereas a score of +3 for TP53 mutation and +2 for other factors associated with an adverse outcome (SF, DNMT3A, WT1 and RUNX1 mutations, older age, higher WBC counts at diagnosis and unfavorable cytogenetics).The algebraic summation of these scores of each patient was the final score.This score system divided the AML patients into five groups with different clinical outcomes (P < 0.001 for both OS and DFS).The 12 patients without chromosome data were not included in the analysis. ", "section_name": "Association of sF mutations with cytogenetic abnormalities", "section_num": null }, { "section_content": "The interaction of SF mutations with mutations of 18 other genes was shown in Table 3.Among the 54 patients with SF mutations, 49 (90.7%)showed additional molecular abnormalities at diagnosis (Tables 1 and3 and Figure 1).Eleven had one additional change, 21 had two, 12 had three, four had four and one had five.Patients with SF mutations had significantly higher incidences of RUNX1, ASXL1, IDH2 and TET2 mutations than those without the mutation (31.5% vs. 10.1%,P < 0.0001; 27.8% vs. 7.8%, P < 0.0001; 20.4% vs. 9.9%, P = 0.0344 and 27.8% vs. 11.4%;P = 0.0022, respectively).The interaction of mutations in each SF gene and other genetic alterations was shown in Supplementary Table 4. ", "section_name": "Association of sF mutations with other molecular gene abnormalities", "section_num": null }, { "section_content": "Of the 363 AML patients undergoing conventional intensive induction chemotherapy, 284 (78.5%) patients achieved a CR.Mutations in any of SF3B1, SRSF2 and U2AF1 were associated with lower CR rates (22.2% vs. 79.7%,P = 0.0005; 45.5% vs. 79.3%,P = 0.0162; 33.3% vs. 79.8%,P = 0.0009; respectively, Supplementary Table 2A, B, C).With a median follow-up of 55 months (ranges, 1.0 to 160), patients with mutations of either SF3B1 or U2AF1 had significantly shorter OS (2 months vs. 29.5 months, P < 0.001 and 4.5 months vs. 26 months, P = 0.001, respectively, Supplementary Figure 1A,E) and DFS (0 month vs. 9 months, P < 0.001 and 0 month vs. 9 months, P < 0.001, respectively, Supplementary Figure 1B,F), while patients with SRSF2 mutation had a significantly inferior OS (14.5 month vs. 29.5 months, P = 0.021, Supplementary Figure 1C) and a trend of shorter DFS than those without the mutation (0 month vs. 9 months, P = 0.172, Supplementary Figure 1D).As mutations of all three individual SF implicated a poor response to treatment and inferior outcome, we therefore analysed the clinical relevance of SF mutations as a whole.Patients with SF mutations had significantly poorer OS and DFS than those without SF mutation (median, 6 months vs. 38 months, P < 0.001, and median, 0 month vs. 10 months, P < 0.001, respectively, Figure 2A,2B).The prognostic differences remained similar among the patients with non-M3 AML (median, 6 months vs. 25 2C and median, 0 month vs. 7.5 months, P < 0.001, Figure 2D, respectively).The same were also true for the subgroup of 161 patients with normal karyotype (median, 4.5 months vs. 61 months, P < 0.001, Figure 2E and median, 0 month vs. 10 months, P < 0.001, Figure 2F, respectively). In multivariate analysis (Table 4), the independent poor risk factors for OS were older age >50 years, higher white blood cell (WBC) counts >50,000/μL, unfavorablerisk cytogenetics and mutations of SF (RR 2.243, 95% CI 1.380-3.647,P = 0.001), TP53, RUNX1, WT1 and DNMT3A.On the other hand, CEBPA double-mutation and NPM1 mutation in the absence of FLT3-ITD (NPM1 + /FLT3-ITD -) were independent favorable prognostic factors.There was a trend of better OS in patients with IDH2 mutation (RR 0.539, 95% CI 0.284-1.020,P = 0.058).Similarly, the independent poor risk factors for DFS included older age > 50 years, higher WBC counts >50,000/μL, unfavorablerisk cytogenetics, and SF, TP53, RUNX1, WT1 and DNMT3A mutations.On the other hand, NPM1 + /FLT3-ITD -was an independent favorable prognostic factor.In the 229 patients with intermediate-risk cytogenetics, the SF mutation was still an independent poor prognosis for OS and DFS (RR, 2.999; 95% CI, 1.002-2.999,P = 0.049 and RR, 1.705; 95% CI, 1.028-2.827,P = 0.039, respectively, Supplementary Table 5). Intriguingly, among the 97 patients receiving allogeneic HSCT, either in first CR (n = 45) or beyond (n = 52), the poor prognostic impact of SF mutation on OS and DFS was lost (P = 0.439 and P = 0.348, respectively).It seems that HSCT may ameliorate the poor survival impact of SF mutations, similar to RUNX1 mutations.[12,13] However, because the number of patients who had SF mutations and received HSCT was limited in our cohort, further studies in more patients are needed to clarify this point. To better stratify the AML patients into different risk groups, a scoring system incorporating SF mutations with ten other prognostic factors, including age, WBC counts, cytogenetics at diagnosis, NPM1/FLT3-ITD, and mutations of CEBPA, IDH2, TP53, DNMT3A, RUNX1 and WT1, into survival analysis was formulated based on the results of our Cox proportional hazards model.The weight of the each variable was based on the value of relative risk (Table 4).To simplify the clinical utilization, a score of -3 was assigned for NPM1 + /FLT3-ITD -and -2 for CEBPA double-mutation and IDH2 mutation whereas a score of +3 for TP53 mutation and +2 for other factors associated with an adverse outcome (SF, DNMT3A, WT1 and RUNX1 mutations, older age, higher WBC counts at diagnosis and unfavorable cytogenetics).The algebraic summation of these scores of each patient was the final score.This score system divided the AML patients into five groups with different clinical outcomes (P < 0.001 for both OS and DFS, Figure 3). ", "section_name": "Impact of sF mutation on response to therapy and clinical outcome", "section_num": null }, { "section_content": "SF mutations were serially studied in 489 samples from 163 patients, including 11 patients with SF mutations and 152 patients without the mutation at diagnosis (Table 5).Among the nine patients with SF mutations who obtained a CR and had available samples for study, eight lost the original mutation at remission status, but one (patient 22) retained it (Table 5).In addition to U2AF1 mutation, patient 22 also harbored concurrent mutations of NRAS and IDH2 at diagnosis and these two mutations disappeared at CR.The amplitude of the mutant sequence of U2AF1 in this patient was much lower at CR compared to that at diagnosis and relapse (Supplementary Figure 2).It implied that the cells with the mutation were present as a minor population at remission suggesting that the mutation was not hereditary, but acquired, and such residual leukemia cells would then cause relapse. In the seven patients who had available samples for serial study at relapse, the original SF mutation could be detected at relapse in six patients (patients 3, 8, 14, 22, 26 and 34), but was lost in one (SRSF2 mutant in patient 36).Because direct sequencing might not be sensitive enough to detect low level of SF mutation signal, we therefore sequenced TA clones of the PCR product from patient 36 at relapse.The original SRSF2 mutant could be detected in one out of 45 clones.Interestingly, acquisition of novel mutations was noted at relapse in three SF-mutated patients (patients 3, 8 and 14, Table 5).On the other side, among the 152 patients who had no SF mutation at diagnosis, none acquired SF mutation at relapse. ", "section_name": "sequential studies of sF mutations", "section_num": null }, { "section_content": "Most studies on SF mutations in AML were focused on small patients cohorts.[2,8,9,11] To the best of our knowledge, this study recruited the largest cohort of de novo AML.Patients with antecedent hematological diseases, family history of myeloid neoplasms or therapy-related AML were excluded the same way we did previously.[14,15] We found that SF mutation was associated with distinct clinic-biological features and was a poor prognostic factor in AML patients, independent of age, WBC counts, karyotype and other genetic markers. Mutations of the SF genes were identified in 54 (10.8%) patients, most commonly in those with intermediate-risk cytogenetics (13.2%).Similar to the data in MDS, the majority of mutations occurred in hotspot areas: K666N and K700E in SF3B1, S34 and Q157 in U2AF1 and P95 in SRSF2.The incidence of SF mutations in AML varied from 4.5%-12.5% in different reports.[2,[8][9][10][11] Yoshida et al found SF3B1, U2AF1 and SRSF2 mutations in 2.6%, 1.3% and 0.7%, respectively, of 151 AML patients.[2] Kihara et al reported 4.5% of 197 patients harbored SF mutations, including SF3B1 (1.5%), U2AF1 (1.5%), SRSF2 (1%) and ZRSR2 (0.5%) mutations.By analyzing the mutations in eight hotspots of SF genes in 325 patients, Taskesen et al showed 1.8% of AML patients had mutations in SF3B1, 1.2% in U2AF1 and 4.6% in SRSF2.[10] In a cohort of 200 adult AML patients reported by the Cancer Genome Atlas (TCGA), the incidence of mutations in 21 spliceosome genes detected by either whole-genome sequencing or wholeexome sequencing was 12.5%; among them, SF3B1 mutation was found in 0.5%, U2AF1 mutation in 4% and SRSF2 mutation in 0.5%.[11] Surprisingly, mutations in SRSF2 gene occurred in 81% of AML patients with isolated trisomy 13. [16] The reason of the variability in the incidence of SF mutations in different studies is unknown but may be due to differences in ethnic background, patient population selected (age range, FAB subtypes and karyotype, etc), the regions of SF genes screened, and the methods used.We analyzed exons 14-15 in SF3B1 genes, exons 2 and 6 in U2AF1 genes and exon 2 in SRSF2 gene to avoid missing some mutations outside hotspot regions.A higher frequency of SRSF2 mutations in this study might be partially due to age effect; elder patients were also enrolled in this cohort and SRSF2 mutation is closely associated with older age in myeloid neoplasm.[17] Although a close association was observed between SF mutations and mutations in certain genes, especially those related to epigenetic modifications, in MDS (such as SF3B1 mutation with DNMT3A mutation, SRSF2 mutation with mutations of RUNX1, IDH and ASXL1 genes and U2AF1 mutation with mutations of ASXL1 and DNMT3A), [4,6,18,19], little is known about the interaction between SF mutations and other molecular genetic alterations in AML patients.In a study of mutational status of three SF genes (SF3B1, U2AF1 and SRSF2), NPM1, FLT3, CEBPA, IDH1, DNMT3A, ASXL1 and NRAS/KRAS in 344 patients, including 47 refractory anemia with excess blasts (RAEB), 29 AML with low BM blast count and other AML patients, Taskesen et al could not find any molecular association. [10] However, with the help of combined genome-wide mRNA expression and DNA-methylation profiling they identified two distinct patient clusters highly enriched for SF-mutated RAEB/AML.One cluster was associated with erythroid phenotype; the other was correlated with NRAS/KRAS mutation (10 out of 25 patients, 40%).However, the reason why these two clusters were defined only by combined genome-wide mRNA expression and DNA-methylation profiling was unclear.In this study, we found SF mutations rarely occurred alone; 49 (90.7%) of 54 patients with SF mutations showed additional molecular abnormalities at diagnosis.This finding is in agreement with the concept that the development of AML requires concerted cooperation of different molecular genetic alterations.[11,20] Intriguingly, patients with SF mutations had significantly higher incidences of RUNX1, ASXL1, IDH2 and TET2 mutations than those without the mutation, similar to the findings in MDS.[4,6,18,19] To the best of our knowledge, this study is the first to evaluate the dynamic change of SF mutation during disease progression in a large cohort of patients with de novo AML.In contrast to the instability of FLT3-ITD during disease evolution, [21] we found that the SF mutation seemed rather stable, analogous to DNMT3A mutations [14,22] At relapse, the original SF mutations in all seven SF-mutated patients studied were retained, but the mutant level in one of them was much reduced at the time of AML relapse as it could only be detected by a sensitive cloning technique, but not by direct sequencing.(patient 36, Table 5) On the other side, among the 152 patients who had no SF mutation at diagnosis, all remained germline of the genes during clinical follow-ups.Taken together, these findings suggest that SF mutations were quite stable during disease evolution and may play an important role in development, but not progression of AML. Few studies regarding the prognostic relevance of SF mutations in de novo AML have been reported.In a study of Taskesen et al, only one distinct SF-mutant patient cluster enriched for NRAS/KRAS mutation (cluster 3, 7.3% of 344 patients) had poorer prognosis.Patients with isolated trisomy 13 reported by Herold et al, in whom high frequencies of mutations in SRSF2 (81%) and RUNX1 (75%) were noted, had a dismal outcome.[16] In this study, we distinctly identified that SF mutation was an important prognostic factor, independent from all other variables in both total cohort and patients with intermediate-risk cytogenetics.Although SF3B1 mutations have been shown to predict better OS in MDS patients, [3,19,23,24] we found the mutation was associated with a lower CR rate (Supplementary Table 2A) and shorter survival in de novo AML patients (Supplementary Figure 1A,B).The reason why SF3B1 mutation has different impact on clinical outcome between patients with MDS and AML remains to be explored.In fact, the reports concerning the prognostic impact of SF3B1 mutation in MDS showed inconsistent and conflicting results.[3,19,23,24] The good prognostic impact of SF3B1 mutation could not be demonstrated in MDS patients in some studies.[3,19,23,24] It was suggested the close association of SF3B1 mutation with old age and DNMT3A mutation and different treatment regimens might influence the implication of this mutation on survival of MDS patients.[19,24] In AML, Lindsley et al [25] first showed that SF mutations as well as ASXL1, EZH2, BCOR, and STAG2 mutations were highly specific for secondary AML, and were secondary-type mutations in therapy-related AML and elderly de novo AML that defined a distinct subgroup of patients with poor outcome.In this study, we only recruited de novo AML patients, the same cohort as we reported previously.[14,15] Secondary AML patients were carefully excluded and SF mutations in this study were closely associated with intermediate-risk cytogenetics, but not poor-risk cytogenetics or complex karyotype, which is frequently seen in secondary AML.The findings from this study reflected the poor prognostic implication of SF mutations in de novo AML patients. Intriguingly, the poor prognostic impact of SF mutation in OS and DFS was lost if the patients received allogeneic HSCT.In other words, HSCT may ameliorate the poor survival impact of SF mutations.Further studies in more patients are needed to clarify this point.To better stratify AML patients into different risk groups, a survival scoring system incorporating SF mutation and ten other prognostic factors, including age, WBC counts, cytogenetics, NPM1/FLT3-ITD, CEBPA, IDH2, RUNX1, WT1, DNMT3A and TP53 mutations, into survival analysis was formulated.Indeed, this scoring system was more powerful than single marker to separate patients into different prognostic groups.Further studies in independent cohorts are needed to validate the clinical implication of the proposed scoring system. There was one potential flaw and limitation in this study.We did not analyze the mutations of all 21 spliceosome genes; the results we obtained might only reflect the clinical relevance of mutations in the three SF genes we analyzed.However, SF3B1, U2AF1 and SRSF2 mutations are the most frequent SF mutations in myeloid neoplasms and can be easily detected by Sanger's sequencing.[2,11] The finding that mutations in these three SF genes predict poor prognosis suggests routine test of these mutations may be helpful in the clinical management of AML patients. In summary, this study demonstrated that SFmutated patients had specific clinic-biologic features and cytogenetic changes.SF mutations were closely associated with RUNX1, ASXL1, IDH2 and TET2 mutations.Furthermore, the SF mutation was an independent poorrisk factor for OS and DFS among total cohort and patients with intermediate-risk cytogenetics.Incorporation of SF mutation with ten other prognostic factors into survival analyses can better stratify AML patients into different risk groups.Sequential study during the clinical course showed that SF mutations were quite stable during AML evolution.These mutations can be potential targets for novel therapies and biomarkers for disease monitoring. ", "section_name": "dIscussIon", "section_num": null }, { "section_content": "From March 1995 to Dec 2008, a total of 500 adult patients with newly diagnosed de novo AML according to the French-American-British (FAB) criteria at the National Taiwan University Hospital (NTUH) were enrolled as previously described.[14,15] Patients with antecedent hematological diseases, history of cytopenia, family history of myeloid neoplasms or therapy-related AML were excluded.Among them, 363 (72.6%) patients received standard induction chemotherapy (Idarubicin 12 mg/m 2 per day on days 1-3 and Cytarabine 100 mg/m 2 per day on days 1-7) and then consolidation chemotherapy with 2-4 courses of high-dose Cytarabine (2000 mg/ m 2 q12h days 1-4, total 8 doses), with or without an anthracycline (Idarubicin or Novatrone), after achieving complete remission (CR).[14,15] The patients with acute promyelocytic leukemia (M3 subtype) received concurrent all-trans retinoic acid and chemotherapy.The remaining 137 patients received palliative therapy due to underlying comorbidity or based on the decision of the patients.Forty-five patients received allogeneic hematopoietic stem cell transplantation (HSCT) in first CR and 52 in relapse/refractory status or second CR or beyond.This study was approved by the Institutional Review Board of the NTUH; and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. ", "section_name": "MAterIAls And Methods subjects", "section_num": null }, { "section_content": "Chromosomal analyses were performed as described previously.[26] ", "section_name": "cytogenetics", "section_num": null }, { "section_content": "A panel of monoclonal antibodies to myeloid associated antigens, including CD13, CD33, CD11b, CD15, CD14, and CD41a, as well as lymphoid-associated antigens, including CD2, CD5, CD7, CD19, CD10, and CD20, and lineage nonspecific antigens HLA-DR, CD34, and CD56 were used to characterize the phenotypes of the leukemia cells as previously described.[14] ", "section_name": "Immunophenotype analysis", "section_num": null }, { "section_content": "Mutation analysis of SF genes, including SF3B1, SRSF2 and U2AF1, was performed by polymerase chain reaction (PCR) and direct sequencing.[17][18][19] Abnormal sequencing results were confirmed by at least two repeated analyses.Sequential analysis of SF mutations during the clinical course was performed in 489 samples from 163 patients.Mutation analyses of 18 other relevant molecular marker genes, including Class I mutations, such as FLT3/ITD and FLT3/TKD, [27] NRAS, [28] KRAS, [28] JAK2, [28] KIT [29] and PTPN11 [29] mutations and Class II mutations, such as CEBPA [30] and RUNX1 [13] mutations, as well as NPM1, [31] WT1, [32] TP53 [33] and those genes related to epigenetic modification, such as MLL/PTD, [34] ASXL1, [35] IDH1, [36] IDH2, [37] TET2 [38] and DNMT3A [14] mutations were performed as previously described.To detect SF mutations at diagnosis, we used DNA amplified in vitro from patients' BM cells by Illustra TM GenomiPhi V2 DNA amplification kit as described by the manufacturer (GE Healthcare, Buckinghamshire, UK).All the mutations detected in such samples were verified in the original non-amplified samples. ", "section_name": "Mutation analysis", "section_num": null }, { "section_content": "For the patients with discrepancy of the mutation status of the SF genes in paired samples, Taq polymeraseamplified (TA) cloning was performed in the samples without detectable mutant by direct sequencing as previously described.[28] ", "section_name": "tA cloning analysis", "section_num": null }, { "section_content": "The discrete variables of patients with and without SF mutation were compared using the chi-square tests, but if the expected values of contingency tables were smaller than 5, Fisher exact test was used.If the continuous data were not normally-distributed, Mann-Whitney U tests were used to compare continuous variables and medians of distributions.To evaluate the impact of SF mutation on clinical outcome, only the patients who received conventional standard chemotherapy, as mentioned above, were included in analysis.[14,15] OS was measured from the date of first diagnosis to the date of last follow-up or death from any cause, whereas relapse was defined as a reappearance of at least 5% leukemic blasts in a BM aspirate or new extramedullary leukemia in patients with a previously documented CR. [39] Disease-free (DF) status indicated that the patient achieved CR and did not relapse by the end of this study.Cox regression survival estimation was used to plot survival curves and to test the difference between groups.Multivariate Cox proportional hazard regression analysis was used to investigate independent prognostic factors for OS and DFS.The proportional hazards assumption (constant hazards assumption) was examined by using Time-Dependent Covariate Cox regression before conducting multivariate Cox proportional hazard regression.The variables including age, WBC counts, karyotype, NPM1/ FLT3-ITD, CEBPA, IDH2, WT1, RUNX1, ASXL1, DNMT3A and TP53 mutations were used as covariates.Those patients who received HSCT were censored at the time of HSCT in survival analysis to ameliorate the influence of the treatment.[14,15] A P-value < 0.05 was considered statistically significant.All statistical analyses were performed with the SPSS 19 (SPSS Inc., Chicago, IL, USA) and Statsdirect (Cheshire, England, UK). ", "section_name": "statistical analysis", "section_num": null } ]	[ { "section_content": "The authors declare no competing financial interests. This paper has been accepted based in part on peerreview conducted by another journal and the authors' response and revisions as well as expedited peer-review in Oncotarget. ", "section_name": "AcKnoWledgMents/grAnt suPPort", "section_num": null }, { "section_content": "", "section_name": "AcKnoWledgMents/grAnt suPPort", "section_num": null }, { "section_content": "", "section_name": "Authors' contributions", "section_num": null }, { "section_content": "The authors declare no competing financial interests. ", "section_name": "conFlIcts oF Interest", "section_num": null }, { "section_content": "This paper has been accepted based in part on peerreview conducted by another journal and the authors' response and revisions as well as expedited peer-review in Oncotarget. ", "section_name": "editorial note", "section_num": null } ]
10.1038/s41598-023-37216-8	Data mining on identifying diagnosis and prognosis biomarkers in head and neck squamous carcinoma	<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background: </jats:bold>Head and neck squamous carcinoma (HNSC) is cancer with a poor prognosis that induces high cancer-related death worldwide. The biomarker screening on diagnosis and prognosis is of great importance. This research is aimed to explore the specific diagnostic and prognostic biomarkers for HNSC through bioinformatics analysis.<jats:bold>Results: </jats:bold>The mutation and dysregulation data were acquired from UCSC Xena and TCGA databases. The top ten genes with mutation frequency in HNSC were <jats:italic>TP53</jats:italic> (66%), <jats:italic>TTN</jats:italic> (35%), <jats:italic>FAT1</jats:italic> (21%), CDKN2A (20%), MUC16 (17%), CSMD3 (16%), PIK3CA (16%), NOTCH1 (16%), SYNE1 (15%), LRP1B (14%). A total of 1,060 DEGs were identified, with 396 up-regulated and 665 downregulated in HNSC patients. Patients with lower expression of <jats:italic>ACTN2</jats:italic> (<jats:italic>P</jats:italic>=0.039, HR=1.3), <jats:italic>MYH1</jats:italic> (<jats:italic>P</jats:italic>=0.005, HR=1.5), <jats:italic>MYH2</jats:italic> (<jats:italic>P</jats:italic>=0.035, HR=1.3), <jats:italic>MYH7</jats:italic> (<jats:italic>P</jats:italic>=0.053, HR=1.3), and <jats:italic>NEB</jats:italic> (<jats:italic>P</jats:italic>=0.0043, HR=1.5) exhibit longer overall survival time in HNSC patients. The main DEGs were further analyzed by pan-cancer expression and immune cell infiltration analyses. We can observe that MYH1, MYH2, and MYH7 were dysregulated in the cancers. Compared with HNSC, their expression levels are lower in the other types of cancers. All the five DEGs have a significant positive correlation with CD4+ T cells and macrophages.<jats:bold>Conclusion: </jats:bold><jats:italic>ACTN2</jats:italic>, <jats:italic>MYH1</jats:italic>, <jats:italic>MYH2</jats:italic>, <jats:italic>MYH7</jats:italic>, and<jats:italic> NEB</jats:italic> genes were significantly associated with poor prognosis in HNSC. Moreover, <jats:italic>MYH1</jats:italic>,<jats:italic> MYH2</jats:italic>, and <jats:italic>MYH7</jats:italic> were expected to be the specific diagnostic and prognostic molecular biomarkers of HNSC.</jats:p>	[ { "section_content": "Mutations in HNSC patients.The mutation profile of HNSC patients from the TCGA database was obtained.Figure 1A showed that the frequency distribution of different variant types was divided into seven according to the impact on protein-coding.We found that missense mutation accounts for the majority of the variant types.In these variant types, the number of SNPs is significantly more than that of INS and DEL (Fig. 1B).Besides, C > T transversion was the primary type of single nucleotide variants (SNV) in HNSC (Fig. 1C).We show genes with a mutation frequency of top25 in Fig. 1D.The top ten genes with a mutation frequency were TP53 (66%), TTN (35%), FAT1 (21%), CDKN2A (20%), MUC16 (17%), CSMD3 (16%), PIK3CA (16%), NOTCH1 (16%), SYNE1 (15%), LRP1B (14%). Differentially expression genes calculation.In these patients, the expression levels in two groups were compared, and a total of 1061 DEGs were identified, with 396 up-regulated and 665 downregulated (Fig. 2A,B).Then, we retained the DEGs with more than 1% mutation frequency.After the screening, 187 common DEGs were obtained (Fig. 2C), including 66 up-regulated and 121 down-regulated. PPI and key module analyses on DEGs.PPI analysis was performed on the common DEGs with more mutation frequency.The PPI network showed 144 nodes and 489 interaction pairs (Fig. 3).The DEGs with more than one connection with other DEGs were retained (Supplementary File 1).In addition, two sub-network modules were screened, with 15 nodes and 102 interaction pairs in module A (score = 14.571), and 14 nodes and 75 interaction pairs in module B (score = 11.538). In addition, we listed the key DEGs in sub-network modules in Table 1. ", "section_name": "Results", "section_num": null }, { "section_content": "We further analyzed the function of DEGs by GO-BP and KEGG.We found that the DEGs in module A are mainly involved in GO-BP terms, including extracellular matrix organization, extracellular structure organization, collagen fibril organization, endodermal cell differentiation, endoderm formation, endoderm development, formation of the primary germ layer, cellular response to amino acid stimulus, collagen-activated tyrosine kinase receptor signaling pathway, and collagen-activated signaling pathway (Fig. 4A).They are also involved in KEGG pathways of protein digestion and absorption, ECM-receptor interaction, AGE-RAGE signaling pathway in diabetic complications, amoebiasis, focal adhesion, human papillomavirus infection, PI3K-Akt signaling pathway, Relaxin signaling pathway, small cell lung cancer, and platelet activation (Fig. 4B).The DEGs in module B mainly participated in muscle contraction, muscle system process, myofibril assembly, cellular component assembly involved in morphogenesis, striated muscle cell development, cardiac muscle cell development, cardiac cell development, muscle filament sliding, actin-myosin filament sliding, and cardiac muscle fiber development (Fig. 4C).In addition, KEGG pathways of hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac muscle contraction, thyroid hormone signaling pathway, adrenergic signaling in cardiomyocytes, cGMP-PKG signaling pathway, viral myocarditis, and arrhythmogenic right ventricular cardiomyopathy were enriched (Fig. 4D).The yellow circle is the down-regulated gene, the blue prism is the up-regulated gene, the node size is based on the degree value, the higher the degree value, the larger the node.lated in HNSC patients (P < 0.05; Fig. 5).Patients with lower expression of ACTN2 (P = 0.039, HR = 1.3),MYH1 (P = 0.005, HR = 1.5),MYH2 (P = 0.035, HR = 1.3),MYH7 (P = 0.053, HR = 1.3), and NEB (P = 0.0043, HR = 1.5) exhibit longer overall survival time.These results demonstrated that these DEGs might be essential for predicting the overall survival of HNSC. ", "section_name": "Function analysis on DEGs in sub-network modules.", "section_num": null }, { "section_content": "Then, we evaluated their expression in other cancers (Fig. 6A-E).ACTN2 was significantly downregulated in bladder urothelial carcinoma (BLCA), kidney chromophobe (KICH), kidney renal papillary cell carcinoma (KIRP), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA), and uterine corpus endometrial carcinoma (UCEC) (P < 0.001); up-regulated in invasive breast carcinoma (BRCA), cholangiocarcinoma (CHOL), and colon adenocarcinoma (COAD).We can observe that although MYH1, MYH2, and MYH7 were dysregulated in some of the cancers, their expression levels are very low, except for HNSC.Therefore, MYH1, MYH2, and MYH7 can be considered specific HNSC biomarkers.As for NEB, it was significantly downregulated in BRCA, HNSC, KICH, KIRP, and THCA; up-regulated in BLCA, CHOL, COAD, esophageal carcinoma (ESCA), liver hepatocellular carcinoma (LIHC), LUAD, LUSC, rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), and UCEC (P < 0.05; P < 0.01; P < 0.001).Moreover, we evaluated the expression levels of ACTN2, MYH1, MYH2, MYH7, and NEB in HPV-positive and -negative HNSC samples.Their expression levels showed no significant differences in HPV-positive and negative HNSC samples (Fig. 6F). ", "section_name": "Key DEG expression validation, survival curve, and UALCAN", "section_num": null }, { "section_content": "The correlation between key DEGs and immune infiltrate cells in total HNSC, HNSC-positive, and HNSC-negative samples was evaluated.As for total HNSC samples, we found that ACTN2, MYH1, MYH2, and MYH7 were negatively correlated with B cell, CD8+T cell, and positively correlated with CD4+T cell, macrophage, neutrophil, and dendritic cell (Fig. 7A-E).All five DEGs have a significant positive correlation with CD4+T cells and macrophages.ACTN2 had positive correlation with CD4+T cell (partial correlation = 0.253; P = 2.03e-08), macrophage (partial correlation = 0.181; P = 6.03e-05), neutrophil (partial correlation = 0.157; P = 5.82e-04), and dendritic cell (partial correlation = 0.161; P = 4.02e-04) in HNSC samples.It showed an opposed correlation in HNSC-HPV positive and HNSC-HPV negative in immune cell infiltration of B cell and CD8+T cell, but not significant (Fig. 7A).Therefore, the HPV infection had no significant influence on immune cell infiltration of ACTN2. MYH1 showed positive correlation with CD4+T cell (partial correlation = 0.201; P = 8.71e-06), macrophage (partial correlation = 0.146; P = 1.31e-03), neutrophil (partial correlation = 0.095; P = 3.76e-02), and dendritic cell (partial correlation = 0.104; P = 2.30e-02; Fig. 7B).The MYH1 showed a consistent correlation in the immune cells of HNSC-HPV positive and negative samples, except in neutrophils.Although MYH1 in HNSC-HPV positive sample is negatively correlated with neutrophils, but is not significant. As for MYH2, MYH7, and NEB were significantly positively correlated with CD4+T cell (P < 0.001; Fig. 7C-E).They showed consistent correlation in the immune cells among the total HNSC, HNSC-HPV positive and negative samples; Some slight correlation opposition was observed in HNSC-HPV positive or negative with total HNSC, but not significant.Therefore, we concluded that no significant differences were identified in HNSC-HPV positive or negative with total HNSC in terms of immune cell infiltration of ACTN2, MYH1, MYH2, MYH7, and NEB. ", "section_name": "Immune cell infiltration correlation analysis.", "section_num": null }, { "section_content": "HNSC, a dreadful opponent, has become one of the world's most deadly diseases.Treatment for HNSC is costly, has a long cycle, and is not affordable.Therefore, the precise and early prediction of HNSC outcomes is critical for therapy options and prognosis improvement.Our present study focused on gene mutation and dysregulation expression in HNSC, expecting to identify essential biomarkers for HNSC. In the present study, we explored the landscape of mutation and dysregulation in HNSC patients, showing that TP53, TTN, FAT1, CDKN2A, and MUC16 were the most predominant mutated genes.The mutation identification was consistent with Jiang et al. 14 .Among human genes, TP53 is a critical tumor suppressor gene, with low expression in normal cells and high expression in malignant tumors, regulating cell proliferation, apoptosis, angiogenesis, and DNA repair 15 .Mutated TP53 was found in various cancers and is correlated with reduced O.S. Furthermore; it showed that HNSC patients with TP53 mutations have a bleak prognosis than TP53-wildtype HNSC 16 .Our present study also found that mutated TP53 is observed in HNSC. Then, the dysregulated genes were identified, with 187 DEGs having more mutation sites.We conducted the PPI analysis on these DEGs and identified two modules.We further analyzed these DEGs in these modules by GO-BP and KEGG analyses.The DEGs in module A are involved in the PI3K-Akt signaling pathway, a classic intracellular signaling pathway regulating cell apoptosis, proan essential pathway that regulates cell apoptosis, prognosis, and metastasis in HNSC [17][18][19] .The DEGs in module B participated in the cGMP-PKG signaling pathway.In addition, Tuttle et al. 20 demonstrated that the cGMP-PKG signaling pathway is one of the therapeutic targets of HNSC. The DEGs of ACTN2, MYH1, MYH2, MYH7, and NEB in module B were significantly associated with O.S. in HNSC.ACTN2 is a member of the spectrin gene superfamily that includes varying groups of cytoskeletal proteins.In breast cancer patients, mutated ACTN2 was related to invasive ductal carcinoma and suggested a worse O.S. than ductal carcinoma in situ 21 .Sun et al. 22 demonstrated that ACTN2 is one of the hub genes selected by bioinformatics methods in PTEN mutation prostate cancer.Xu et al. 23 revealed that negative ACTN2 expression contributed to a better O.S. in HNSC patients, consistent with our present study.Therefore, ACTN2 might be an essential biomarker for predicting O.S. of HNSC, which might be employed in clinical. Through pan-cancer analysis, we found that MYH1, MYH2, and MYH7 are dysregulated in cancers, including HNSC.However, compared with HNSC, they were lower expressed in other cancers, revealing that these three genes can be considered single HNSC biomarkers.So far, different mutations in multiple members of the MYH gene family are associated with human hereditary diseases 24 .Among them, MYH2 mutations can cause a class of skeletal muscle diseases characterized by ophthalmoplegia.MYH7 mutations can cause skeletal muscle diseases, including myosin deposition myopathy, and Distal Laing myopathy is also closely related to hypertrophic cardiomyopathy.However, MYH1, MYH2, and MYH7 functions in HNSC are rarely mentioned.Further study into the interaction of the MYH gene family and HHSC appears to be an intriguing research topic. As we all know, HPV infection is the most common causative factor for HNSC.Considering the significant influence of HPV on HNSC, some molecular research on HNSC have divided the samples into HNSC-HPV positive and negative groups.For example, Zhang et al. 25 found that Dedicator of cytokinesis 8 (DOCK8) is identified as one prognosis biomarker associated with immune infiltration HPV-positive HNSC.Our present study identified five prognosis DEGs that are significantly related to the O.S. in HNSC.To determine whether the HPV infection had significant influences on the results.We further analyzed their expression in HPV-positive and -negative HNSCs, and no significant differences were observed.Moreover, we also tested the immune cell infiltration of DEGs in both HPV-positive and -negative HNSCs.Although studies have demonstrated that HPV influences the immune infiltration situation 26 , it is not observed in ACTN2, MYH1, MYH2, MYH7, and NEB.As genes highly expressed in the tumor cells are expected to affect tumor purity positively, the association between the expression of prognosis DEGs and six immune infiltrates was evaluated.We found that all five DEGs have a significant positive correlation with CD4+T cells and macrophages.Therefore, we speculate that genetic mutations and differential expression of ACTN2, MYH1, MYH2, MYH7, and NEB genes in HNSC cells may be important drivers of CD4+T cell and macrophage infiltration. ", "section_name": "Discussion", "section_num": null }, { "section_content": "In our present study, we identified the biomarker of HNSC in terms of diagnosis and prognosis.TP53 was found as the most mutated gene in HNSC.ACTN2, MYH1, MYH2, MYH7, and NEB genes were significantly associated with poor prognosis.Moreover, MYH1, MYH2, and MYH7 were expected to be the specific diagnostic and prognostic biomarkers molecular biomarkers of HNSC. ", "section_name": "Conclusions", "section_num": null } ]	[ { "section_content": "This work was supported by the General Program of National Natural Science Foundation of China (81972555 to Fang-Zhou Liu). ", "section_name": "Funding", "section_num": null }, { "section_content": "The RNA-Seq and mutation data re-analyzed during the current study are available in the TCGA database with the links of https:// xenab rowser.net/ datap ages/?datas et= TCGA-HNSC.htseq_ fpkm.tsv& host= https% 3A% 2F% 2Fgdc.xenah ubs.net and https:// xenab rowser.net/ datap ages/?datas et= TCGA-HNSC.varsc an2_ snv.tsv& host= https% 3A% 2F% 2Fgdc.xenah ubs.net. ", "section_name": "Data availability", "section_num": null }, { "section_content": "Data collection.The clinical data and gene expression profiles of HNSC patients were downloaded from the UCSC Xena database (https:// xenab rowser.net/ datap ages/).As calculated, a total of 506 tumors and 44 adjacent HNSC samples were included in our present study.The gene expression levels of examples were provided in Supplementary File 2. The somatic mutation data was from the TCGA database by Varscan 27 . Screening of high-frequency mutation genes.The maftools 28 was used to summarize the HNSC mutation information downloaded from the TCGA official website and screen the high-frequency mutation genes.The top 25 high-frequency genes with the mutation frequency were exhibited with a waterfall chart. Differentially expressed gene (DEG) identification.The DEGs in tumor tissues were identified according to the FPKM value by limma package 29 (Version 3.10.3),using P-value < 0.05 and log 2 fold change (F.C.) > 2 as thresholds.The Pheatmap R package was employed for drawing the heatmap and volcano. Then, the common DEGs with high-frequency mutation genes that exhibit a mutation frequency of more than 1% were retained for further study. The PPI analysis was performed on all the common DEGs using STRING 30 (Version: 10.0, http:// www.string-db.org/) database, with a 0.4 (medium confidence) parameter.The network was constructed by Cytoscape (version: 3.2.0) 31.The most significant clustered modules in the PPI network were analyzed using the Cytoscape plug-in MCODE 32 (Version 1.4.2,http:// apps.cytos cape.org/ apps/ MCODE) method, with a threshold of score ≥ 10. The biological process (B.P.) of Gene Ontology (G.O.) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on DEGs were performed by clusterProfiler 33 , with adjusted P-value < 0.05 and gene number count ≥ 2. The top ten G.O. terms and KEGG pathways were visualized. The key DEGs in the module were subjected to expression verification and survival analysis by GEPIA 34 .Survival analysis was grouped by median data and performed using OS survival information.The pan-cancer expression levels of key DEGs significantly associated with prognosis were assessed using UALCAN (http:// ualcan.path.uab.edu/ index.html).The expression levels of key DEGs in HNSC-positive and HNSC-negative samples were evaluated. As for the immune cell infiltration analysis, the HNSC samples were divided into HNSC-positive and HNSC-negative.Then, the infiltration of the key DEGs in six immune cells (B cells, CD 4+ T cells, CD 8+ T cells, neutrophils, macrophages, and dendritic cells) were identified through the TIMER database. Ethics approval and consent to participate.UCSC Xena and TCGA belongs to public databases.The patients involved in the database have obtained ethical approval.All methods were carried out in accordance with relevant guidelines and regulations. G.J. all data analysis, and writing; Z.Y. data analysis, and writing; Y.Z.performed part of the analysis; X.Z.conceptualization, interpretation and discussion of results; F.L. conceptualization, advised all research, design of experiments, evaluation and discussion of results, revision of manuscript, and provided research funding.All authors have read and agreed to the published version of the manuscript. The authors declare no competing interests. ", "section_name": "Methods", "section_num": null }, { "section_content": "Data collection.The clinical data and gene expression profiles of HNSC patients were downloaded from the UCSC Xena database (https:// xenab rowser.net/ datap ages/).As calculated, a total of 506 tumors and 44 adjacent HNSC samples were included in our present study.The gene expression levels of examples were provided in Supplementary File 2. The somatic mutation data was from the TCGA database by Varscan 27 . Screening of high-frequency mutation genes.The maftools 28 was used to summarize the HNSC mutation information downloaded from the TCGA official website and screen the high-frequency mutation genes.The top 25 high-frequency genes with the mutation frequency were exhibited with a waterfall chart. Differentially expressed gene (DEG) identification.The DEGs in tumor tissues were identified according to the FPKM value by limma package 29 (Version 3.10.3),using P-value < 0.05 and log 2 fold change (F.C.) > 2 as thresholds.The Pheatmap R package was employed for drawing the heatmap and volcano. Then, the common DEGs with high-frequency mutation genes that exhibit a mutation frequency of more than 1% were retained for further study. ", "section_name": "Methods", "section_num": null }, { "section_content": "The PPI analysis was performed on all the common DEGs using STRING 30 (Version: 10.0, http:// www.string-db.org/) database, with a 0.4 (medium confidence) parameter.The network was constructed by Cytoscape (version: 3.2.0) 31.The most significant clustered modules in the PPI network were analyzed using the Cytoscape plug-in MCODE 32 (Version 1.4.2,http:// apps.cytos cape.org/ apps/ MCODE) method, with a threshold of score ≥ 10. ", "section_name": "Protein-protein interaction (PPI) and module analyses.", "section_num": null }, { "section_content": "The biological process (B.P.) of Gene Ontology (G.O.) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on DEGs were performed by clusterProfiler 33 , with adjusted P-value < 0.05 and gene number count ≥ 2. The top ten G.O. terms and KEGG pathways were visualized. ", "section_name": "Functional analysis.", "section_num": null }, { "section_content": "The key DEGs in the module were subjected to expression verification and survival analysis by GEPIA 34 .Survival analysis was grouped by median data and performed using OS survival information.The pan-cancer expression levels of key DEGs significantly associated with prognosis were assessed using UALCAN (http:// ualcan.path.uab.edu/ index.html).The expression levels of key DEGs in HNSC-positive and HNSC-negative samples were evaluated. ", "section_name": "Key DEG expression validation and survival curve analysis.", "section_num": null }, { "section_content": "As for the immune cell infiltration analysis, the HNSC samples were divided into HNSC-positive and HNSC-negative.Then, the infiltration of the key DEGs in six immune cells (B cells, CD 4+ T cells, CD 8+ T cells, neutrophils, macrophages, and dendritic cells) were identified through the TIMER database. Ethics approval and consent to participate.UCSC Xena and TCGA belongs to public databases.The patients involved in the database have obtained ethical approval.All methods were carried out in accordance with relevant guidelines and regulations. ", "section_name": "Immune cell infiltration analysis.", "section_num": null }, { "section_content": "G.J. all data analysis, and writing; Z.Y. data analysis, and writing; Y.Z.performed part of the analysis; X.Z.conceptualization, interpretation and discussion of results; F.L. conceptualization, advised all research, design of experiments, evaluation and discussion of results, revision of manuscript, and provided research funding.All authors have read and agreed to the published version of the manuscript. ", "section_name": "Author contributions", "section_num": null }, { "section_content": "The authors declare no competing interests. ", "section_name": "Competing interests", "section_num": null } ]
10.1186/s13046-021-02115-1	Restoration of the immune function as a complementary strategy to treat Chronic Lymphocytic Leukemia effectively	<jats:title>Abstract</jats:title><jats:p>Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy characterized by uncontrolled proliferation of B-cells and severe immune dysfunction. Chemo(immuno)therapies (CIT) have traditionally aimed to reduce tumor burden without fully understanding their effects on the immune system. As a consequence, CIT are usually associated with higher risk of infections, secondary neoplasms and autoimmune disorders. A better understanding of the biology of the disease has led to the development of therapeutic strategies which not only act against malignant B-cells but also reactivate and enhance the patient’s own anti-tumor immune response. Here, we review the current understanding of the underlying interplay between the malignant cells and non-malignant immune cells that may promote tumor survival and proliferation. In addition, we review the available evidence on how different treatment options for CLL including CIT regimens, small molecular inhibitors (i.e, BTK inhibitors, PI3K inhibitors, BCL-2 inhibitors) and T-cell therapies, affect the immune system and their clinical consequences. Finally, we propose that a dual therapeutic approach, acting directly against malignant B-cells and restoring the immune function is clinically relevant and should be considered when developing future strategies to treat patients with CLL.</jats:p>	[ { "section_content": "Chronic Lymphocytic Leukemia (CLL) is the most common B-cell malignancy in the Western world [1].CLL is a lymphoproliferative disease characterized by the accumulation of mature monoclonal B-cells with a typical immunophenotype (i.e, CD5+CD23+ and other B-cell markers) which accumulate in peripheral blood, bone marrow and lymph nodes.A hallmark of the pathophysiology of CLL is the dysfunction of the immune system which is mostly translated in a state of humoral and cellular immunodeficiency and higher prevalence of autoimmune disorders [2,3]. In CLL, tumor cells influence the immune system to escape immunosurveillance and create an immunosuppressive microenvironment.In general, there are four mechanisms used by CLL cells to escape from the control of immune cells: (1) non-immunogenic tumor cell death; (2) expansion and recruitment of immunosuppressive cells, including T regulatory (T-reg) cells, M2 macrophages and myeloid-derived suppressor cells (MDSC); (3) depletion and/or inhibition of antitumor immune cells such as Th1 T-cells and CD8+ cells; and (4) production of immunosuppressive soluble factors such as IL-10 and TGF-β [4].These changes are highly relevant for the maintenance and progression of the tumor and contribute to the complex manifestations of the disease.Recently, it has been reported that immune microenvironment plays a significant role in progression of the disease in contrast to clonal evolution [5]. At the end of the 90´s, the introduction of chemotherapy in combination with anti-CD20 monoclonal antibodies such as rituximab was an important step forward in CLL therapy and the outcome of patients significantly improved.However, most patients will eventually relapse.Importantly, CIT decreases the disease burden but, at the same time, exacerbates certain defects of an already dysfunctional immune system which further impacts patients' health, with increased infection morbidity and secondary neoplasms [6].The introduction of small molecule inhibitors, such as Bruton tyrosine kinase (BTK) [7][8][9][10], phosphoinositide 3-kinase (PI3K) [11], or B-cell Lymphoma 2 (BCL-2) [12,13], has changed the treatment paradigm by providing even more effective therapeutic options for CLL patients. Immunotherapeutic strategies (immune checkpoint inhibitors, bi-/tri-specific antibodies, chimeric antigen receptor (CAR)-T-cells) are emerging as relevant treatment options in many malignancies by reverting or bypassing immunosuppression caused by the tumor, but the results have been disappointing so far in CLL.Effective restoration of the immune competence of a patient should have anti-tumor effects that may help control the disease. There is accumulating evidence of the relevance of the interaction between CLL B-cells and the tumor microenvironment.Restoring autologous T-cell responses remains a potential therapeutic option in CLL, since it should promote anti-tumor immunity without the GvHD (graft-versus-host disease) complications of allogenic transplants [14].Here, we review the current treatment options (Fig. 1) in CLL in the context of the new molecular understanding of the immune system and its interactions with this neoplasm. ", "section_name": "Background", "section_num": null }, { "section_content": "Immune dysfunction is a fundamental characteristic of CLL that can be present even in early stages of the disease.However, these immunologic aspects have been a deprioritized side in the research of this field for many years.The clinical consequences are an increased risk of secondary malignancies [15] and infections, the latter being one of the leading causes of mortality among patients with CLL [16].Conversely, in some cases, the perturbation of the immune regulation can result in Fig. 1 Current relevant therapies in the treatment of patients with CLL that are discussed in this review and main molecular targets.Ab, antibody; BCL-2, B-cell lymphoma 2; BTK, Bruton's Tyrosine kinase; CAR-T, chimeric antigen receptor T; Fc, fragment crystallizable region; PI3K, phosphoinositide 3-kinase; ROR1, Receptor Tyrosine Kinase Like Orphan Receptor 1; scFv, single-chain variable fragment autoimmune cytopenia in CLL [17,18].In this section, we will review the causes and consequences of the immune dysregulation that characterizes CLL. ", "section_name": "Pathophysiology of the Immune System in CLL", "section_num": null }, { "section_content": "CLL B-cells have shown to have immunosuppressive effects on normal T and B-cells by direct cell-to-cell contact and by tumor-derived soluble factors [6]. It has been proposed that CLL B-cells may resemble natural B-reg cells [6] since CLL B-cells also secrete IL-10, which contributes to their immunosuppressive function, and inhibit TNF-α secretion by macrophages.In addition, IL-10 can have autocrine deleterious effects by promoting proliferation and differentiation of CLL B-cells.Clinically relevant, increased IL-10 levels have been associated with a diminished survival in CLL patients [19]. Moreover, a high number of T-reg cells are observed in CLL, which may negatively impact antitumor immunity provided by tumor-infiltrating lymphocytes (TILs) [20]. Myeloid-derived suppressor cells (MDSCs) are more abundant in CLL patients than in healthy individuals.Tumor-associated macrophages (TAMs) secrete immunosuppressive cytokines (IL-10 and TGF-β).Both MDSCs and TAMs promote expansion of T-reg cells and inhibit activation of T-cell surveillance providing a suppressive microenvironment that allows CLL proliferation [21,22]. The tumor microenvironment is also immunosuppressive in the CLL lymph nodes (LN) with an increased number of tumor-supportive lymphocytes (follicular T helper (Tfh) and T-reg) and fewer cytotoxic ones, compared to peripheral blood.CD8+ T-cells, in addition, have an increased expression of the inhibitory receptors PD-1 that limits their potential to build an immune response in the long term due to stable epigenetic changes difficult to revert [23,24]. ", "section_name": "Immunosuppressive environment in CLL", "section_num": null }, { "section_content": "Defects in the innate immune system (Fig. 2) have been reported in CLL patients: the phagocytic function and chemotaxis of neutrophils are reduced [25].Circulating monocytes have gene expression patterns related to deregulation of phagocytosis and inflammation, potentially due to effects of CLL-derived soluble inhibitory molecules [26].Similarly, NK (natural killer) cells are increased but have impaired production of cytotoxic granules and reduction of cytolytic molecules releases [27].In addition, low expression of activating molecules in the surface of CLL B-cells adds to the already poor function of NK cells [15]. Adaptive immunity is also dysfunctional, with hypogammaglobulinemia that is exacerbated with disease progression and possibly involving all immunoglobulin classes.In recent studies, a direct correlation has been observed between low levels of IgG and IgA and increased mortality due to bacterial infections [6]. Regarding T-cell immunity, the absolute numbers of CD4+ and CD8+ T-cells are higher in patients with CLL than in age-matched healthy donors but not proportionally higher since the ratio CD4+/CD8+ is reduced.Interestingly, the reduction of CD4+/CD8+ ratio has been shown to correlate with disease progression.Also, the impaired function of the main subsets of T-cells has been described including both CD4+ T helper (Th) cells and CD8+ T cytotoxic lymphocytes cells with defects in cytoskeletal formation and cytotoxic activity [28]. ", "section_name": "Defects in the innate and adaptive immune systems", "section_num": null }, { "section_content": "The T-cell compartment dysregulation in CLL was described a few decades ago; however, the role of T-cells in the pathogenesis of CLL is still not fully understood.CLL B-cell expansion occurs in parallel to an uncontrolled T-cell proliferation, but it is unclear if the increase is the natural response of the immune system to CLL or if it is an active player of the disease. Cell to cell communication is also impaired in the CLL environment: synapse dysfunction was identified in T-cells that have been in contact with CLL B-cells inducing T-cell anergy [29]; PD-1+ T-cell frequency is increased in progressive CLL which together with chronic low-affinity self-antigen exposure induces a state of \"pseudo-exhaustion\" [30]. Additional perturbations of T-cell subsets have been described and one of the most interesting is the disruption between Th1 and Th2 balance (Fig. 3).Th1 cells help keep malignant cells under control by the release of molecules such as IL-2 and IFN-γ; on the other hand, Th2 cells protect malignant cells by promoting B-cell antibody production and interfering with cytotoxic T-cells [31].In healthy conditions, this system helps maintaining normal immune system homeostasis, but it is altered in CLL patients. Naïve CD4+ T-cells differentiation towards Th1, Th2 or Th17 cells is controlled by interleukine-2-inducible kinase (ITK), among other molecules.T-cell receptor (TCR) activation (especially low affinity interactions with the major histocompatibility complex (MHC) I) triggers a signal cascade activating ITK and promoting Th2 differentiation.Th2 produce cytokines such as IL-4, IL-5 and IL-13, that trigger positive feedback for proliferation of Th2 cells and cross-inhibit other Th lineages.In CLL, the equilibrium is skewed towards Th2 differentiation, inhibiting effective anti-tumor responses and promoting CLL B-cell proliferation [32].Recent evidence showed the relevance of another subtype of T-cells important for anti-tumor activity: γδ T-cells can identify and kill tumor cells in a MHC-independent manner and recognize a broader spectrum of neoplastic antigens, but these cells are also affected by the immunosuppressive environment of CLL [33]. ", "section_name": "Impaired anti-tumor T-cell response", "section_num": null }, { "section_content": "", "section_name": "Current treatment approaches and impact in the immune function", "section_num": null }, { "section_content": "Purine analogs such as fludarabine, cladribine, or pentostatin, and alkylating agents such as bendamustine, chlorambucil or cyclophosphamide, interfere with both tumor and healthy dividing cells.As a consequence, chemotherapy reduces CLL B-cell numbers, but also decreases other healthy cells including T-cells and slows down the recovery to normal levels, which ultimately leads to infections [34,35]. Two frequently used CLL regimens until recently are fludarabine/cyclophosphamide/rituximab (FCR) and bendamustine/rituximab (BR).For many years, FCR has been the standard of care in previously untreated young and fit CLL patients.FCR resulted in high overall response rates with a complete responses of 44%, and median progression-free survival (PFS) of 51.8 months [36]; the benefit was primarily driven by low-risk patients with mutated IGHV and without 17p deletion [37].However, this combination is considered too toxic for frail and/or elderly patients due to appearance of hematological toxicities [36].Fludarabine can cause reductions in T-cell numbers, with a higher effect on CD4+ cells [34], γδ [34] and T-reg cells [38], and serious opportunistic T-lymphopenia-associated infections during the first year after FCR treatment, such as Pneumocystis jirovecii and Legionella pneumoniae, pulmonary aspergillosis, endemic fungus infections or disseminated listeriosis [39].Also, long-term toxicities including risk of secondary hematological malignancies (i.e, acute myeloid leukemia, myelodysplasia) have been associated with the FCR regimen [40].T-cell repertoire renewal after FCR treatment was found to be affected through ablation and immune reconstitution, rather than expansion of previous T-cell clones, thus impairing early antigen recognition [41]. BR has demonstrated efficacy in CLL although not as significant as FCR [42].This regimen is frequently used in patients unsuitable for fludarabine-based treatments.It is associated with fewer hematological complications and infections compared FCR, but reductions of CD4+ cells, delays in recovering normal CD4+ cells levels and infections were common [35]. Anti-CD20 antibodies (rituximab and obinutuzumab) target CLL B-cells for complement-dependent cytotoxicity (CDC), direct cell death, antibodydependent cell-mediated phagocytosis (ADCP) or antibody-dependent cellular cytotoxicity (ADCC) by NK cells.Therefore, even though CLL B-cells express low levels of the necessary ligands to activate NK cytolytic activity, anti-CD20-coated CLL cells are able to trigger an ADCC response [15].It has been reported that rituximab treatment may reduce humoral response to \"recall antigens\", which may be due to the depletion of healthy antibody-producing B-cell clones, although how this may affect the rate of infections or secondary malignancies is not clear yet [43]. Obinutuzumab, a type II humanized anti-CD20, has shown different properties from rituximab, displaying superior ADCC and direct cell death activity, similar ADCP but inferior CDC in vitro.Obinutuzumab caused significant and sustained reductions of CD4+ cells after one infusion.In addition, a reduction of CD8+ cells was also detected but levels were normalized after 6 months [44].These effects were observed 24-72h post-infusion while depletion of B-cells suffered no significant reduction until later.One more effect in the immune system was the early and sustained reduction in NK cells [44]. Therefore, immune-related risks associated with anti-CD20 therapy are infections, for example, hepatitis B reactivation, which has been described in patients with CLL and other B-cell lymphomas [45]. ", "section_name": "Chemoimmunotherapy", "section_num": null }, { "section_content": "BTK belongs to the Tec family of kinases and plays a role in immune cell signaling.BTK is part of the BCR (B-cell receptor) signaling pathway that promotes survival and proliferation of malignant B-cells.BTK inhibitors, including ibrutinib, acalabrutinib and zanubrutinib have shown clinical efficacy in patients with CLL [7,[46][47][48]. BTK inhibitors produce transient lymphocytosis caused by the mobilization of B-cells from LN, bone marrow and spleen to peripheral blood.They also produce changes in the tumor environment due to a decrease in the expression of immunosuppressive molecules such as PD-L1, IL-10, CD200 or BTLA in CLL B-cells [49,50]. Interestingly, another related Tec kinase, ITK, is also inhibited by some of these small molecules.ITK is an important component of TCR signaling and promotes Th2, T-reg, and Th9 CD4+ cells differentiation and reduces cytotoxic CD8+ and CD4+ Th1 cells differentiation [31].This is relevant for tumor control since skewing T-cell differentiation towards a Th2 response would create a pro-tumor environment and cause a decrease of Th1 functions in tumor surveillance.Therefore, the inhibition of ITK also has an impact on the T-cell compartment [31]. Ibrutinib elicits changes in the tumor microenvironment that both control CLL B-cells proliferation and reestablish immune surveillance (Fig. 4).Regarding the T-cell compartment, ibrutinib causes a transient increase of CD4+ and CD8+ T-cells numbers, mostly effector and effector memory T-cells [50], and is associated with an expansion in the TCR repertoire diversity [51].In the long term, ibrutinib causes a decrease in pathologically high circulating B-cells and exhausted and chronically activated T-cells but preserved naïve T-cells and NK cells [52].Ibrutinib also shifts the equilibrium of CD4+ T-cells from Th2 and Th17 cells to a Th1 environment by inhibiting ITK, reverses CD8+ cell exhaustion [53] and favors activation of CD8+ cytotoxic T-cells [31].Moreover, it has been observed that ibrutinib-based therapy improves functional immune and cytolytic synapses between T-cells and CLL B-cells compared to FCR treatment, and enhances the polarization of perforin towards CLL B-cells in T-cell synapses [41], helping to restore the cytotoxic capacity of T-cells to kill tumor cells [54]. In addition, ibrutinib is associated with a the reduction of the T-reg/CD4+ cells ratio, the net increase of Th17 cells and the inhibition of the expression of PD-1 and CTLA-4 in CD4+ and CD8+ T-cells all together leading to a reduction in the immunosuppressive status and an improvement in immune surveillance [50,55]. Ibrutinib also modulates the function of other immune cells, such as macrophages, by reducing the cell-cell contacts with CLL B-cells in the bone marrow, migration and nitric oxide production [55,56], and of monocytes, by impairing differentiation towards M1 macrophages, causing adhesion and phagocytic deficiencies and increasing the immunosuppressive profile of nurse-like cells [57]. These changes in the immune system triggered by the treatment with ibrutinib have positive clinical relevance; for example, the restoration of TCR diversity has been shown to correlate with clinical efficacy and lower infection rates [31,51,53]; and the restoration of the CD4+/ CD8+ ratio may help improve pre-existing autoimmune cytopenias and decrease the rate of emergent events [18]. In summary, ibrutinib effectively controls CLL via a dual mechanism.First, it has shown to act directly on CLL B-cells, reducing the number of malignant cells and their immunosuppressive effects and second, through modulation of the microenvironment and restoration of the physiological immune function, by increasing T-celldependent immune surveillance.With the available evidence up to date, ibrutinib is the first therapy to show that a strategy to address tumor burden and immune function simultaneously could improve the disease management (Fig. 4).Moreover, this offers significant potential to combine with other therapeutic options. Acalabrutinib is a selective inhibitor of BTK that has shown efficacy in CLL [46,58].Since ITK binding is essentially absent for acalabrutinib, the effects on Th2 to Th1 switch in the CD4+ T-cell compartment are not observed [50].Similarly to ibrutinib, a reduction of immunosuppressive molecules such as CD200, BTLA, PD-1 and CTLA-4 expression, together with a decreased production of IL-10, have been noted [50].Acalabrutinib has some other effects on the immune system, for example, it impairs efficient responses on macrophages (M1 polarization and TNF-α secretion) and neutrophils (phagocytosis, oxidative burst and others), important for antimicrobial infection control [59]. Zanubrutinib is another highly specific BTK inhibitor with low ITK activity that has shown potential in CLL [48].It has shown significant reductions on PD-1 and CTLA-4 expression and a decrease in T-reg cells; but no changes in Th1/Th2 ratio [60]. ", "section_name": "Novel Agents BTK inhibitors", "section_num": null }, { "section_content": "PI3K-δ is mostly expressed in hematopoietic cells and frequently overexpressed in B-cell lymphomas.It plays a major role in B-cell signaling and has also been involved in T-reg cells and MDSC function [11].Two Pi3K inhibitors have been approved, idelalisib and duvelisib).Idelalisib is a PI3K-δ selective inhibitor with efficacy in CLL by inhibiting CLL B-cell proliferation, survival, adhesion, and homing [11]. Idelalisib has also effects on non-B-cells: it decreases the number of T-reg cells, although this effect was observed mostly in patients with hepatotoxicity [61], and impairs their differentiation and suppressive functions [20].In addition, it decreases the production of cytokines and other factors such as TNF-α, CD40L and IL-6 by T-cells; IFN-γ by NK cells and IL-10 by T-reg cells [20]. The reduction of T-reg cells and soluble pro-tumor factors makes idelalisib an interesting candidate for combination with immunotherapies but it is not frequently considered an option because those additional effects have been associated with relevant immune-related toxicities of concerning severity and frequency, such as hepatotoxicity or enterocolitis [61]. ", "section_name": "Pi3K inhibitors", "section_num": null }, { "section_content": "BCL-2 is an anti-apoptotic factor that is overexpressed in CLL.Venetoclax is a BCL-2-selective BH3-mimetic that has been effective in treating patients with CLL, by directly inducing apoptosis of CLL B-cells [12]. Venetoclax has also effects on the T-cell compartment, reducing the total number of CD4+ and CD8+ cells although this is thought to be an indirect effect of the decrease of the tumor.Among the surviving cells, there was a high proportion of CD4+ and CD8+ effector memory T-cells while the proportion of naïve T-cells was lower, these remaining cells kept their proliferation capacity intact [62].In addition, venetoclax reduces the immunosuppressive environment by reducing the number of Tfh cells, T-reg cells and PD-1+ CD8+ T-cells, as well as the overproduction of inflammatory cytokines [62]. Moreover, some studies suggest venetoclax reduces the number of NK cells but restoration of their function has been observed after treatment [63]. ", "section_name": "BCL-2 inhibitors", "section_num": null }, { "section_content": "CAR-T cells are T-cells from a patient that are modified ex vivo and reintroduced to target cancer cells.Infusion of ex-vivo manipulated cells may reduce the influence of the immunosuppressive tumor microenvironment and, conversely, the use of patient's own cells should reduce the risk of GvHD [14].Antigens of choice for initial CAR-T cells were the surface antigens CD19, CD20 and CD23, causing also a reduction in healthy B-cells.Overall, either because of lack of efficacy or the development of adverse events, CAR-T cells have not reached clinical expectations in CLL [64,65], at least partially due to the innate dysfunction in T-cells coming from patients from which the CAR-T cells are produced.Interestingly, therapies that restore immune competency, such as ibrutinib, may enhance the efficacy of CAR-T cells [63].A similar approach is being studied with CAR-NK cells, and preliminary results suggest better results may be obtained in CLL [66]. Immune Checkpoint Inhibitors (ICI) reactivate immune response towards tumors by blocking inhibitory signals mediated by PD-1 and CTLA-4.ICI have transformed the treatment paradigm in many solid tumors, but their efficacy remains suboptimal in CLL.PD-(L)1 pathway is active in CLL but the expression of PD-L1 in CLL B-cells is usually low.In recent trials, the efficacy of anti-PD-1 monotherapy has been moderate in CLL with Richter transformation (RT), and limited in CLL without RT [67].Some studies have shown that addition of ibrutinib to the therapy with ICI may improve its efficacy, especially in patients with RT [68,69]. Another strategy arising in oncology is the development of Bi/Tri-specific antibodies.These molecules are able to force the interaction between tumor and cytotoxic cells and their domains can be combined to target different molecules, for example, CD19, CD20 or ROR1, in B-cells and CD3 in effector T-cells, although other combinations and different effector cells (NK cells) have been used as well [2]. CD19/CD3-scFv-Fc bi-specific antibodies are able to recruit autologous T-cells to kill malignant cells and have shown potential in treating CLL.For example, it has been observed that blinatumomab is able to mediate CLL B-cells and cytotoxic T-cells interaction and trigger tumor cell death in vitro but no activity has been detected in vivo [70].Interestingly, these effects may be faster in samples of patients treated with some BTK inhibitors [71].Similarly, ROR1xCD3 bi-specific antibodies had cytotoxic activity against CLL B-cells, but when autologous T-cells were used, only those isolated from patients previously treated with ibrutinib had a significant effect [72]. ", "section_name": "Immunotherapies", "section_num": null }, { "section_content": "Considering the potential of the different therapies discussed above on the restoration of the T-cell compartment and other components of the immune system, it is reasonable to try different combinations that, at least theoretically, could improve the patient outcomes by leveraging the patient's own immune system response (Table 1). ", "section_name": "Effects of treatment combinations on the CLL immune environment", "section_num": null }, { "section_content": "BTK inhibitors have been studied in combination with anti-CD20 antibodies.BTK inhibitors can inhibit anti-CD20-induced NK cell cytokine secretion, cell degranulation and NK ADCC in vitro [73] but still, these combinations have demonstrated preserved activity of anti-CD20 antibodies clinically.In fact, similar or superior PFS has been observed between acalabrutinib + obinutuzumab vs acalabrutinib alone [58].Other studies have shown positive synergistic effects when ibrutinib is combined with obinutuzumab [8]. Impact of idelalisib combinations in CLL and specifically in the T-cell compartment has been studied to a lesser extent.Idelalisib has similar effects to ibrutinib when tested in vitro in combination with anti-CD20 antibodies, although inhibition of antibody-dependent Table 1 Available evidence of effects of different therapeutic agents alone or in combination on the immune system in chronic lymphoid leukemia (CLL).cell-mediated effector mechanisms seems to be less pronounced [73]. Studies combining venetoclax and obinutuzumab have shown a decrease not only in CLL B-cells but also healthy B-cells, T-cells and NK cells.Decreases seem to be more profound in Tfh, T-reg and PD-1+ CD8+ T subtypes.In addition, this combination generates a decrease in IFN-γ and TNF-α produced by CD8+ T-cells [23].In clinical trials, venetoclax plus anti-CD20 antibodies combinations have shown durable responses, in some cases even after cessation of treatment, although the effects of these combinations on the immune function need to be further investigated [74]. ", "section_name": "New agents + anti-CD20 antibodies combinations", "section_num": null }, { "section_content": "Numerous reports have shown that ibrutinib enhances the effects of immunotherapies.It has been hypothesized [75] that ibrutinib could restore antitumor T-cell immune response in an ITK-dependent manner and enhance ICI effectiveness.A lymphoma model with an ibrutinib-insensitive BTK version has been developed and it has been observed that anti-PD-L1 treatment is T-cell-dependent and moderately effective but the combination with ibrutinib led to significant improvement in the response, through its effects in the immune function restoration rather than the direct effects in CLL B-cells [75].Two clinical trials have reported data on the combination of nivolumab and ibrutinib.Activity of the combination is limited in CLL patients but is promising in patients with RT [67,69].An additional clinical trial reported results on the use of pembrolizumab in CLL and RT patients, where 4 out of 9 patients with RT had objective responses, and none of the 16 patients with CLL.Interestingly, all 4 patients achieving response were previously treated with ibrutinib [68]. An explanation of these results may be that CLL B-cells and RT-cells may be expressing different tumor neoantigens (or RT-cells may be more efficient presenting them).This causes that only RT-cells neoantigens are identified by T-cells, helping ICI trigger responses against them, executed by ibrutinib-mediated re-activated T-cells [67]. Venetoclax helps keep effector memory T-cells viable and functional, and this may be important to improve the effects of immunotherapies [62], although a decrease in naïve T-cells may reduce the response to tumor-derived neoantigens.So far, there is only preliminary in vitro evidence that venetoclax may have synergistic effects with ICI and therefore further research is needed to confirm this observations [62]. ", "section_name": "New agents + Immunotherapy", "section_num": null }, { "section_content": "There is also evidence that ibrutinib can help enhance the CAR-T cell therapy.A preclinical study showed that co-administration of ibrutinib and CD19-directed CAR-T cells (CTL019) in a CLL mouse model improves the efficacy of the CAR-T cells.Ibrutinib can improve the proliferative capacity of T-cells from CLL patients, and it has been shown that the critical expansion of CTL019 is greater when collected from patients that have undergone at least 5 cycles of ibrutinib treatment.In addition, PD-1 and CD200 inhibitory molecules decrease expression in T and CLL B-cells, respectively.Interestingly, these effects have not been observed when cells are treated in vitro with ibrutinib [63].In clinical trials, ibrutinib-treated patients had significantly better response rates to CAR-T cells compared to ibrutinib-naïve ones, with morphologic complete responses, measurable residual disease negativity, absence of disease in lymph nodes and less severity in cytokine release syndrome [65,76]. Another type of cell therapy involving another subpopulation of T-cells, Vγ9Vδ2-T-cells, has shown potential in CLL.These cells are able to recognize metabolites produced during malignant transformation and trigger a MHC-independent cytotoxic response [33].Similar to other T-cells, Vγ9Vδ2-T-cells function is impaired in CLL, and cell expansion in vitro is inefficient.Information regarding this subpopulation of T-cells is limited but preliminary results suggest that ibrutinib may induce Th1 skewing of these cells in vitro, making it a potential combination strategy for autologous Vγ9Vδ2 T-cell therapy [33]. Idelalisib has been shown to improve autologous CAR-T cell generation, expansion and cytotoxic effects when isolated from idelalisib-treated patients or when incubated with idelalisib.It promotes enrichment of naïve-like T-cells and increase of CD8+ cytotoxic T-cells.It has also been observed that idelalisib causes a decrease in expression of PD-1 [77]. BTK inhibitors have also demonstrated enhancement of bi-specific antibody efficacy as previously explained creating an interesting rationale to combine these agents. ", "section_name": "New agents + cell therapies", "section_num": null }, { "section_content": "Both ibrutinib and venetoclax have been reported to reduce tumor pro-survival Tfh and T-reg cells and reactivate T-cells as monotherapies [50,78].The combination of both molecules has been studied in patients with CLL with results reaching superior complete responses and MRD negativity [79].Ibrutinib enhances venetoclax killing effects by sensitizing CLL B-cells to the BCL-2 antagonism of venetoclax [80]. Regarding effects in the immune environment, research is still ongoing but there is some data from the combination of venetoclax with ibrutinib or obinutuzumab [23].With both venetoclax plus ibrutinib or venetoclax plus obinutuzumab strategies, reductions in healthy B-cells, [23], mediated mostly by venetoclax. ", "section_name": "Other combinations", "section_num": null }, { "section_content": "A decrease in the production of IFN-γ and TNF-α in CD8+ T-cells has only been observed in the venetoclax plus obinutuzumab combination while a decrease in IL-4 production by CD4+ T-cells has only been observed in the venetoclax plus ibrutinib combination, consistent with the restoration of the Th1/Th2 balance described for ibrutinib [23].Other relevant differences are that an improvement in NK cell function has only been observed in the venetoclax plus obinutuzumab combination while a trend of improvement in the antibody production has only observed in the venetoclax plus ibrutinib combination [23]. CLL is a heterogeneous disease caused by dysregulation at multiple levels.Current treatment options are not curative and complex combinations with multiple drugs are being tested to reduce the residual disease in a durable way and improve anti-tumor response by the immune system.Targeting various disrupted pathways at the same time seems a reasonable option to increase the efficacy and reduce relapses, but careful consideration must be taken because the molecular pathways leading to disease and the off-targets effects are not fully understood.Key factors to consider when designing combination studies are (1) targeting different molecular pathways with scientific evidence of synergistic effects, (2) using treatments with non-overlapping safety profiles, (3) designing the treatment regimen to minimize undesired effects and (4) including a significant set of biomarkers that allows the interpretation of the results. Examples of such complex regimes are ibrutinib plus obinutuzumab plus venetoclax, designed to maximize the efficacy keeping adverse events low.Positive results have been recently confirmed in a phase 2 trial [81], showing deep responses, and an acceptable safety profile.Various phase 3 cooperative group studies are currently ongoing (NCT04608318, NCT03701282 and NCT03737981).Other combinations still need further investigation. ", "section_name": "NK cells, and T-cells, specifically a reduction of Tfhcells, T-reg cells and PD-1+ CD8+ T-cells are observed in vitro", "section_num": null }, { "section_content": "Despite recent progress on treatments for patients with CLL, progressive disease is still frequent in many cases.Due to selective pressure in the presence of therapies, resistant clones may proliferate and cause the disease to progress.Targeted therapies are especially prone to resistance with relatively simple mutations in the target protein or pathway, but thanks to methodological advances, we are starting to understand much more complex signaling networks that can create forms of resistance beyond classic genetics.Single-cell technologies, immune-phenotyping, single-cell transcriptome profiling, functional studies, and chromatin mapping, all integrated with bioinformatic analytic tools, may help understand the supportive role of the immune microenvironment in CLL progression and the co-evolution of cells involved in the disease, directly or indirectly [5,41,52,[82][83][84]. Mutations in BTK and PLCG2, the kinase immediately downstream of BTK, have been described as common mutations in patients progressing on ibrutinib [85,86].Interestingly, these resistant clones would provide protection to neighboring cells with wildtype BTK [87], and they were detectable many months before clinical progression [88].Resistance to ibrutinib can go beyond the BTK pathway, and it has been described for MCL that tumor microenvironment provides initial drug resistance through interactions with the malignant cells through integrin β1-ILK [89] and through multiple other signaling networks in CLL, identified by patient-specific signatures [83]. Similarly, mutations in BCL2, detectable months earlier than clinical progression using droplet digital PCR, have been shown to decrease the efficacy of venetoclax [90].Activated T-cells produce IL-4 and IL-21 that can stimulate the CD40-CD40L interaction and increase the expression of the anti-apoptotic factor BCL-xL, among other factors [91]. One of the most frequently used strategies to avoid proliferation of resistant clones is combining different targeted therapies.Combination of ibrutinib and venetoclax would reduce the probabilities of proliferation of clones with resistant mutations in both pathways simultaneously.Additionally, it has been shown that microenvironment cytokines (IL-10, sCD40L or CpG-ODNs) can significantly reduce the synergy of the combination in an NF-κB-dependent manner [91,92]. In general, the immune microenvironment creates protective conditions through a network of complex signal interactions to resist therapeutic interventions.Hypoxia has been identified as key condition on setting a protective niche for CLL cells.Under hypoxia, macrophages acquire a tumor-permissive M2 phenotype, T-cells become immunosuppressive and MDSC increase presence [93,94] directly impacting cell survival and drug resistance.Ibrutinib-induced apoptosis, for example, is reduced under these conditions [95], but at the same time, ibrutinib treatment can counteract, at least partially, the establishment of the protective microenvironment, by suppression of NAMPT (factor involved in linking oxygen use, metabolism, and immune function) transcription, important for the differentiation of tumor-supporting M2 macrophages [96].A key mediator of the hypoxia effects is HIF-1, found to be overexpressed in CLL [95] and it has been shown that inhibition of HIF-1 with BAY87-2243 has synergistic effects when combined with other agents, such as ibrutinib [95]. Interestingly, hypothesis-generating studies have shown that impairing the microenvironment can help improve efficacy of targeted therapies in general.Ascorbic acid is a pro-oxidant agent that has shown to overcome the protective effect of the microenvironment.It can potentiate the effects of ibrutinib, idelalisib and venetoclax in vitro and it was able to reduce the viability of CD40L/IL-4-stimulated CLL cells [97]. Even though these results are preliminary, new high throughput and single-cell methods may help understand better the complexity of these interactions and can contribute to design therapeutic combination strategies that target the tumor microenvironment to reduce progression in CLL and identify patterns that are able to predict response to treatment. ", "section_name": "Role of the immune system in resistance to treatment", "section_num": null }, { "section_content": "Patients with CLL have a characteristic disruption of the immune function and addressing it in parallel to the tumor burden should be central in the management of the disease.Treating the disease by restoring the antitumor activity of the patient's immune system can lead to sustained and deep responses and reduce side effects and non-progression deaths. CLL treatments may exacerbate immune defects, causing infections, autoimmune diseases or secondary neoplasias [98].Data on these effects are scarce but we are starting to understand the impact of CLL treatments on these side effects: ", "section_name": "Clinical considerations of immune restoration/ Expert insight", "section_num": null }, { "section_content": "Chemoimmunotherapy effects on the T-cell compartment may also lead to infections [16].Idelalisib has been associated with an increase in the risk of Pneumocystis jirovecii pneumonia and other opportunistic infections [61].ICIs have shown multiple examples of immunerelated adverse events in different tumors, requiring immunosuppressive treatment leading to secondary infections [98].Blinatumomab causes hypogammaglobulinemia and venetoclax has been associated with neutropenia and infections [98].A slight increase in bacterial, viral and opportunistic infections has been observed with ibrutinib, caused by defects in the innate immune function, although these infections appear mostly during the first months of therapy.Interestingly, prolonged treatment with ibrutinib restores humoral immunity and immunoglobulin levels [98] and a reduction of infections has been reported when ibrutinib is used beyond 6 months [99].Moreover, ibrutinib may also attenuate the exacerbated immune response in severe infections, such as in the case of visceral leishmaniasis [100] or Staphylococcus aureus [101], or reduce the burden of the inflammatory process during the infection, for example in the case of COVID-19 [102] or pneumococcal pneumonia [103]. ", "section_name": "Infections", "section_num": null }, { "section_content": "Chemoimmunotherapy may impair further the function of effector T-cells, and in some cases, such as fludarabine-based regimens, may increase the risk of autoimmune cytopenias (AIC), although addition of cyclophosphamide appears to decrease the incidence of fludarabine-dependent AIC [17].Idelalisib has shown to reduce T-reg cells, which are key players in the control of autoimmune responses and excessive reactions to non-self-antigens [61].In the case of ibrutinib, data have shown that it causes a reduction of Th17 cells, decreasing the risk of treatment-emergent AICs [17], and providing better control in patients with pre-existing AIC [18]. ", "section_name": "Autoimmune cytopenias", "section_num": null }, { "section_content": "The incidence of secondary malignancies is increased in CLL patients potentially due to the disrupted immune surveillance.CIT regimens such as FCR have been associated with a higher incidence of secondary malignancies, particularly hematological neoplasias [40].This effect has been shown to be exacerbated in patients with prolonged cytopenias, potentially due to the prolonged treatment with rituximab.Recently, in a retrospective unicentric study of patients treated with BTKi in first-line (20% of patients) and in the relapse/refractory setting (60% of patients received alkylating agents), the observed rate of secondary malignancies was 2.2, similar to the reported rate in a large cohort of CLL patients followed at a single tertiary center prior to the use of BTKi [104][105][106].In the CLL14 trial, secondary malignancies seem more frequent in venetoclax combined with obinutuzumab (18%) vs. chlorambucil plus obinutuzumab (10.3%) after a followup of 3 years.Additional follow-up will be needed to confirm this association [13,107]. ", "section_name": "Secondary malignancies", "section_num": null }, { "section_content": "CLL has undergone relevant clinical advances in the past decades, both in the understanding and the treatment of the disease.For many years, the focus of the therapeutic approaches has been to decrease the number of B-cells to reduce the tumor burden.New insights have shown that the interaction of leukemic cells with T-cells and other players of the immune system participate in the pathogenesis of the disease and contribute to a decrease of the overall health status of the patient, but this knowledge has not fully been applied to clinical practice yet.Some real-world studies are starting to look at relevant immune parameters, such as IgA in ibrutinib-treated patients [108] but more information is needed to have a broader picture of the dynamics of the whole immune system in the treated patients.We need to further understand the roles of the different immune cells in the disease to apply therapeutic strategies that not only eliminate malignant B-cells but also restore the immune competence of the patient toward anti-tumor surveillance and immunity. There is growing evidence that T-cells play a very relevant role in the disease progression and understanding how T-and B-cells create a pro-tumor environment is essential to treat CLL patients.CLL B-cells can cause an immunosuppressive effect on T-cells, which exhibit exhaustion features that abrogate immune control of the tumor.A whole pro-tumor strategy is put in place by the immune system: decrease in Th1 cells (anti-tumor) is offset by an increase of Th2 cells (pro-tumor), cytotoxic T-cells number and function are inhibited, immunosuppressive MDSC, T-reg cells and Tfh cells increase their function, inhibitory cytokines (i.e.IL-10) and checkpoint receptors (i.e.PD-1) are upregulated and the conditions in the lymph nodes are even more immunosuppressive. Keeping in mind that not only eliminating malignant B-cells, but also restoring the immune function may have significant positive consequences in the clinical practice, especially in CLL, that immune dysfunction is the cause of most of the complications for this disease.Also, understanding the relevance of the other immune players in CLL and how different treatments affect them would have a direct impact in successfully managing the disease.First, because this will allow us to avoid therapies that negatively impact the immune function, such as some chemotherapies.Second, because it will reduce the number of adverse events related to the immune dysfunction, for example infections, autoimmunity and second malignancies.Third, because this will facilitate the design of combination strategies, not only to reach deeper durable responses but to attack the disease from different fronts, with higher chances of synergistic and long-term effects.And four, it will allow us to successfully use the new generation of therapeutic strategies, most importantly immunotherapies and cell therapies, in CLL, so far inefficacious due to the immune dysfunction in CLL patients. Management of CLL multilaterally may be the best option for patients but still, caution with unexpected effects, and prevention of known adverse events together with prompt management of side effects may help in the practice until a better understanding of the disease and the effects of the different treatments is achieved. There is still need for more research to understand the effects of each treatment option (monotherapies and combinations) in the tumor environment and current technology can yield insightful data.Thus, it is essential to include broader biomarker analysis in clinical studies, that go beyond analysis of B-cells and include T-cell subpopulations and soluble factors at baseline and different timepoints. Moreover, whatever we learn about immunotherapies in such a paradigmatic immunosuppressed disease as CLL can help improve their efficacy in other tumors where immune function is impaired. ", "section_name": "Conclusions", "section_num": null } ]	[ { "section_content": "The authors wish to thank Alberto Moldón, PhD for editorial support. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Janssen-Cilag, S.A. has provided financial aid for editorial and publication fees ", "section_name": "Funding", "section_num": null }, { "section_content": "ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent cell-mediated phagocytosis; AIC: autoimmune cytopenias; BCL-2: B-cell Lymphoma 2; BCR: B-cell receptor; BR: bendamustine/rituximab; BTK: Bruton tyrosine kinase; CAR : chimeric antigen receptor; CDC: complement-dependent cytotoxicity; CIT: Chemo(immuno)therapies; CLL: Chronic Lymphocytic Leukemia; CpG-ODNs: CpG oligodeoxynucleotides; CTL019: CD19-directed CAR-T cells; FCR: fludarabine/cyclophosphamide/rituximab; GvHD: graft-versus-host disease; HCK: hematopoietic cell kinase; ICI: Immune Checkpoint Inhibitors; ILK: integrin-linked kinase; ITK: interleukine-2-inducible kinase; LN: lymph node; MCL: mantle cell lymphoma; MDSC: myeloid-derived suppressor cells; MHC: major histocompatibility complex; NAMPT: nicotinamide phosphoribosyltransferase; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; NK: natural killer; PFS: progression-free survival; PI3K: phosphoinositide 3-kinase; PLCG2: phospholipase C gamma 2; RT: Richter transformation; T-reg: T regulatory cells; TAM: Tumor-associated macrophages; TCR : T-cell receptor; Tfh: follicular T helper; Th: T helper; TILs: tumor-infiltrating lymphocytes. All the authors defined the general structure and content of the manuscript.Dr. Hernández-Rivas and Dr. Villanueva drafted the first version of the article, and Dr. Moreno, Dr. Muñoz and Dr. Terol revised the manuscript and provided additional information.All the authors have read and approved the final manuscript. Carol Moreno is a consultant for Abbvie, Acerta, AstraZeneca, Janssen, Pharmacyclics and Sunesis; received speakers bureau fees from Abbvie and Janssen; and received grant/research support from Gilead, Janssen and Roche.Cecilia Muñoz is a consultant for Janssen.María José Terol is a consultant for Abbvie, Gilead, Janssen, Roche and Takeda; received grant/research support from Gilead and Janssen.José-Ángel Hernández-Rivas is a consultant for Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Abbreviations", "section_num": null }, { "section_content": "ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent cell-mediated phagocytosis; AIC: autoimmune cytopenias; BCL-2: B-cell Lymphoma 2; BCR: B-cell receptor; BR: bendamustine/rituximab; BTK: Bruton tyrosine kinase; CAR : chimeric antigen receptor; CDC: complement-dependent cytotoxicity; CIT: Chemo(immuno)therapies; CLL: Chronic Lymphocytic Leukemia; CpG-ODNs: CpG oligodeoxynucleotides; CTL019: CD19-directed CAR-T cells; FCR: fludarabine/cyclophosphamide/rituximab; GvHD: graft-versus-host disease; HCK: hematopoietic cell kinase; ICI: Immune Checkpoint Inhibitors; ILK: integrin-linked kinase; ITK: interleukine-2-inducible kinase; LN: lymph node; MCL: mantle cell lymphoma; MDSC: myeloid-derived suppressor cells; MHC: major histocompatibility complex; NAMPT: nicotinamide phosphoribosyltransferase; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; NK: natural killer; PFS: progression-free survival; PI3K: phosphoinositide 3-kinase; PLCG2: phospholipase C gamma 2; RT: Richter transformation; T-reg: T regulatory cells; TAM: Tumor-associated macrophages; TCR : T-cell receptor; Tfh: follicular T helper; Th: T helper; TILs: tumor-infiltrating lymphocytes. ", "section_name": "Abbreviations", "section_num": null }, { "section_content": "All the authors defined the general structure and content of the manuscript.Dr. Hernández-Rivas and Dr. Villanueva drafted the first version of the article, and Dr. Moreno, Dr. Muñoz and Dr. Terol revised the manuscript and provided additional information.All the authors have read and approved the final manuscript. ", "section_name": "Authors' contributions", "section_num": null }, { "section_content": "", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "", "section_name": "Not applicable", "section_num": null }, { "section_content": "", "section_name": "Declarations Ethics approval and consent to participate Not applicable", "section_num": null }, { "section_content": "", "section_name": "Consent for publication Not applicable", "section_num": null }, { "section_content": "Carol Moreno is a consultant for Abbvie, Acerta, AstraZeneca, Janssen, Pharmacyclics and Sunesis; received speakers bureau fees from Abbvie and Janssen; and received grant/research support from Gilead, Janssen and Roche.Cecilia Muñoz is a consultant for Janssen.María José Terol is a consultant for Abbvie, Gilead, Janssen, Roche and Takeda; received grant/research support from Gilead and Janssen.José-Ángel Hernández-Rivas is a consultant for ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.1186/s12935-022-02679-8	Potential of antibody–drug conjugates (ADCs) for cancer therapy	<jats:title>Abstract</jats:title><jats:p>The primary purpose of ADCs is to increase the efficacy of anticancer medications by minimizing systemic drug distribution and targeting specific cells. Antibody conjugates (ADCs) have changed the way cancer is treated. However, because only a tiny fraction of patients experienced long-term advantages, current cancer preclinical and clinical research has been focused on combination trials. The complex interaction of ADCs with the tumor and its microenvironment appear to be reliant on the efficacy of a certain ADC, all of which have significant therapeutic consequences. Several clinical trials in various tumor types are now underway to examine the potential ADC therapy, based on encouraging preclinical results. This review tackles the potential use of ADCs in cancer therapy, emphasizing the essential processes underlying their positive therapeutic impacts on solid and hematological malignancies. Additionally, opportunities are explored to understand the mechanisms of ADCs action, the mechanism of resistance against ADCs, and how to overcome potential resistance following ADCs administration. Recent clinical findings have aroused interest, leading to a large increase in the number of ADCs in clinical trials. The rationale behind ADCs, as well as their primary features and recent research breakthroughs, will be discussed. We then offer an approach for maximizing the potential value that ADCs can bring to cancer patients by highlighting key ideas and distinct strategies.</jats:p>	[ { "section_content": "Cancer therapy remains a difficult task.Chemotherapy has a significant clinical benefit for many tumours, but it has low selectivity and high toxic effects, result in devastating effects and decreased therapeutic efficacy [1].Antibody-drug conjugates (ADCs) are a promising cancer treatment that includes delivering toxic drugs to specific tumor cells that exhibit specific antigens connected to malignancy.The antibody, cytotoxic agent, and linker are the three primary structural units of an ADC.ADCs are expected to provide powerful therapeutic modalities against various cancers by combining the selectivity of monoclonal antibodies (mAbs) and the efficacy of various chemotherapeutics [2].Together, the three components comprise a highly effective anti-tumour agent directly and selectively providing chemotherapy drugs to cancer cells, directed by antibodies with exceptional specificity and affinity. Cleavage of the ADCs linker components by certain tumor-associated enzymes (i.e.matrix metalloproteinases) or by lower pH encountered in the tumour microenvironment results in the release of the active component [3].These non-internalizing ADCs did not increase drug selectivity and, as a result, did not reduce toxicity considerably [4].Despite the fact that ADCs have been studied for many years, we have only just recognized their true potential, thanks to significant advancements in linker and conjugation technology, as well as very powerful cytotoxic drugs [5].ADCs are intended to broaden the therapeutic window of these medications by only delivering them to tumour cells that express a specific antigen targeted by the ADC's mAb antigen [6,7].The properties of the antibody, therapeutic payload, and linker are critical in the overall efficacy of ADCs, which is dependent on intricate interactions between the ADCs and numerous tumour cell and tumour microenvironment (TME) targeting components [8]. Despite promising ADC-induced therapeutic activity against resistant and recurrent cancers, several barriers remain to their widespread use, including unidentified drug resistance mechanisms, toxicity, the lack of predictive prognostic biomarkers, and their clinical advantages over standard therapies.The development of new ADCs is a continual process that relies on advancements in several technologies such as biosynthesis of novel linkers, mAb synthesis and manufacturing, and the introduction of new payloads that are more powerful against tumour cells with fewer systemic side effects. ", "section_name": "Introduction", "section_num": null }, { "section_content": "In the twenty-first century, the development of ADCs has reached significant milestones.Since the early 1900s, efforts have been made to improve the safety and efficacy of Paul Ehrlich's \"magic bullet\", which was the first therapeutic technique to convey lethal drugs to selected cancer cells depending on the presence of cell specific antigen(s) [9,10].Leukemia cells were targeted after the successful chemical linkage of polyclonal rodent immunoglobulins and methotrexate [11].Hybridoma technology permitted the manufacturing of mAbs in 1950, and by the early 1970s, it had sparked important breakthroughs in the field of ADCs, both in vitro and in vivo [12]. The use of ADCs in animal models was described in the literature in the 1960s, and clinical trials with ADCs based on mouse immunoglobulin G (IgG) molecules were conducted in the 1980s [13]. The first ADC to be approved by the US Food and Drug Administration (FDA) for the treatment of patients with acute myeloid leukaemia was gemtuzumab ozogamicin (developed by Wyeth). This was followed by the approval of two second generation ADCs: brentuximab vedotin (developed by Seattle Genetics) in 2011 [14,15] and trastuzumab emtansine (also known as T-DM1 and ado-trastuzumab emtansine; developed by Roche) in 2013 [16], both of which target the cancer antigens CD30 (also known TNFRSF8) and human.Since 2013, the field has changed dramatically.More than 30 new ADCs have entered clinical development (all for oncological indications), and more than 60 ADCs are currently in clinical trials [17].A list of the most recent ADCs currently approved by the US FDA is provided by Drago el al [18,19]. Clinical trials for treating cancer patients with ADCs began in 1980, however the trials' clinical usefulness was hampered by the development of medication toxicity without a significant clinical benefit [20][21][22].Gemtuzumab ozogamicin, an FDA approved CD33-targeted medication for the treatment of relapsed and/or refractory acute myeloid leukemia (R/R) has been withdrawn from the market due to unfavorable adverse effects (AE) [23][24][25].Brentuximab vedotin, a CD30-targeted ADC, and ado-trastuzumab emtansine (T-DM1), a HER2-targeted ADC, were approved in 2011 and 2013, respectively, for the treatment of R/R classical Hodgkin lymphoma and trastuzumab-resistant metastatic breast cancer [15,26,27].Several ADCs are currently being studied in preclinical and clinical development, and the FDA has fully approved nine of them [28]. ", "section_name": "History, design, construction and mechanism of action of ADC", "section_num": null }, { "section_content": "ADCs are primarily composed of three major components: a drug, a linker, and an antibody.The efficacy of each ADC is largely determined by differences in the three fundamental components of ADCs.The development and purification of mAbs utilizing proper cell culture techniques are among the phases in the production of antibody-drug conjugates.Chemically generated and refined cytotoxic payloads.After being functionalized using a specific linker, the mAbs are finally attached to the cytotoxic drug payload (Fig. 1). ", "section_name": "Components of ADC: mAb, linker, and payloads", "section_num": null }, { "section_content": "In addition to cancer treatment, antibody-based therapies have made significant advances in the treatment of other diseases including autoimmune diseases, and cardiovascular and bone diseases [29].One of the most crucial parts of ADC design is antibody selection,and high antigen specificity [30].Antibodies with low specificity that cross-react with other antigens might have unpredictable effects, by interacting with healthy tissues, it might cause off-target toxicities or cause premature clearance from the body before it reaches the tumor site [30].Immunoglobulin M (IgM), A, D, IgE, and IgG are the five types of antibodies.Among the five immunotherapy classes, IgG is the most commonly used [31].Despite the enormous potential for innovation offered by antibody fragments and bispecific antibodies, immunoglobulin G (IgG) is currently the most common used in in ADCs [32][33][34].The classical complement system is activated by IgG subclasses, particularly IgG1 and IgG3.The membrane attack complex (MAC) forms pores on the tumour cell surface leading to cancer cell lysis [35].IgG1 antibodies have similar serum half-lives to their IgG2 and IgG4 counterparts, but higher complementfixation and FcR-binding efficiency.Although IgG3 antibodies are the most immunogenic, they are often avoided in ADC design due to their short circulation half-lives [36].The immunogenicity degree of an ADC is a critical aspect that influences circulatory half-life [7].mAbs can penetrate tumor after being administered into the bloodstream [2].The antibody's size, which typically accounts for roughly 95% of an ADC's bulk, prevents ADCs from spreading into tumor tissue. ", "section_name": "Antibodies selection", "section_num": null }, { "section_content": "Searching for cell-surface proteins expressed in tumours rather than non-malignant tissues has been one guiding methodology in selecting the right mAb target [37].HER2, TROP2, and Nectin 4 are effective targets for ADCs now approved for the treatment of solid malignancies [38][39][40].With the exception of a tiny fraction of lymphocytes, CD30 is a target of brentuximab vedotin and is expressed by malignant lymphoid cells in Hodgkin lymphoma and ALCL in the setting of haematological malignancies [41].Similarly, inotuzumab ozogamicin, polatuzumab vedotin, and belantamab mafodotin are highly specific for hematological malignancies lineages [42,43]. Different mAbs may have different Fc-dependent effector activities [44].As a result, mAbs designed for other therapeutic uses may not be the optimal ADC backbones, in particular when considering mounting evidence that internalization and intracellular trafficking of ADC are critical to ADC cytotoxicity.Pertuzumab's affinity for HER2 is pH-dependent, unlike trastuzumab, resulting in rapid dissociation of the Ab-Ag complex in a low-pH environment.As a result of this discovery, a preclinical recombinant pertuzumab-based ADC with increased cytotoxicity was developed [45].Variant proteins are more prone to ubiquitylation, absorption, and/or instability than wild-type counterparts when targeted by ADCs [46,47].ADCs based on mAbs that target proteins with mutations of truncal oncogenic driver (for instance some mutant versions of EGFR) could attain tumour specificity levels hitherto only achieved with extremely selective inhibitors of small-molecule tyrosine kinase [48,49].Bispecific antibodies have opened up new research and development opportunities.Antibody absorption and/or processing, but also tumor selectivity, could all benefit from such compounds [50]. ", "section_name": "mAb target selection", "section_num": null }, { "section_content": "The specificity, efficacy, and safety of an ADC are determined by the design, structure, and chemistry of the linker that connects the cytotoxic payload to the antibody.Linkers are typically designed to be constant in the blood system (enabling for a prolonged timeframe of bloodstream), but labile enough to efficiently deliver the cytotoxic payload to the tumour [51]. Cleavable and non-cleavable linkers are the two types of linkers.Following exposure to acidic or reducing environments or proteolytic enzymes, cleavable linkers are cut and release the ADC's cytotoxic payload (for example, Non-cleavable linkers are becoming more appealing than cleavable linkers due to the advantage of greater plasma stability.Furthermore, studies show that noncleavable linkers perform far better in vivo, with payload release occurring primarily in the lysosome following ADC internalisation and destruction of both the antibody and the linker (Fig. 2).As a result, the danger of systemic toxicity from premature payload release is reduced.As a result, non-cleavable linkers may offer a wider therapeutic window, as well as increased stability and tolerability [53].T-DM1 with mafodotin belantamab is one of two FDA-approved ADCs with non-cleavable linkers [18]. The systemic stability of ADCs post administration is one of the crucial issues to ensure the efficacy of ADCs, and several strategic approaches are taken for overcoming this issue.ADCs should ideally remain stable or intact in the circulation before entering target cells, but there are cases where ADC catabolites are still biologically active [54,55].ADC stability refers primarily to metabolic stability or integrity.To improve ADC stability, several approaches involving conjugation site selection and linker modification have been developed [54].In general, modifications to each component (e.g., antibody, linker, and payload) can be performed for this purpose.The conjugation site, linker length, and linker steric hindrance are effective general approaches for site-specific ADCs and should be more broadly applicable to a variety of ADC platforms [54].By choosing a more sterically hindered conjugation or attachment site, the antibody can provide the desired steric shield.On the other hand, introducing proximal steric hindrance around the cleavable or labile site of the linker has been shown to be an effective method of improving stability [56].ADC biotransformation and drug-antibody-ratio (DAR) profiling have evolved into critical integrated data for assessing and comprehending ADC stability [54,57]. ", "section_name": "Linker design and technologies", "section_num": null }, { "section_content": "Monoclonal antibodies (mAbs) are well-known therapeutic agents used to treat a wide range of illnesses, including cancer [58].Because of the limitations of mAbs' anticancer activity, researchers are working to improve their potential efficacy.These efforts include mAb conjugation to radionuclides, fusion with immunotoxins, and coupling to ADCs.Payload [59] is the combination of a Fig. 2 ADC internalization and destruction of both the antibody and the linker mAb with a cytotoxic agent or a small molecule.Methotrexate, doxorubicin, and vinca alkaloids are examples of traditional chemotherapy drugs with proven anticancer activity [21,60,61], were initially carried by ADCs.ADCs sometimes required high dosages to be effective, as result of increasing systemic toxicities [62].Currently, optimizing ADCs is a never-ending problem, with most research and development activities focusing on the mAb or chemical linker, on small-scale endeavors, aimed to optimize the cytotoxic payload.There is a dearth of diversity in the medicinal payloads used in the 114 finished or continuing human trials, with only 7 payload formulations described (4 additional ongoing clinical studies with undetailed structures).Natural products account for six of the seven payload mixes, emphasizing the importance of natural materials as cytotoxic payloads for ADC in research investigations [63].Furthermore, the findings demonstrate that a small part of the mAbs targeting the tumor (on the order of 0.1 percent) penetrates tumor tissue, emphasizing the significance of larger cytotoxicity payloads for treatment response [64,65].These discoveries contributed to the growth of ADCs, which include highly effective chemotherapeutic medications like as auristatins, calicheamicins, camptothecin, and maytansinoids analogs that can be lethal even at sub-nanomolar quantities [66,67].Nine cytotoxins were generated from plants, and 21 were natural product formulations from 79 anticancer and antiviral approved medications, according to FDA investigation from 1983 to 2002 [68].Furthermore, 13 of the 39 anticancer compounds were based on natural chemicals.Sixty percent of contemporary pharmaceuticals are bioengineered from natural sources [68,69]. For determining ADC efficacy, the drug-antibody ratio (DAR), or the amount of drug molecules attached to a single ADC, is critical.DAR varies a lot and is influenced by other ADC variables [70].The DAR values are also affected by the conjugation site and whether light or heavy conjugated chains are used [70].The DAR value affects the medicine's effectiveness since low drug loading reduces potency, whereas high drug loading can affect toxicity and pharmacokinetics (PK) [71,72].In general, there are two types of payloads that are commonly utilized in ADC design, as listed below. Rapid plasma clearance may limit the ability of smallmolecule drug conjugates to reach tumor cells or poorly vascularized tumors or the central nervous system [73][74][75].Other innovative ADCs include immunostimulatory agents such Toll-like receptor agonists, chemokines, or STING agonists to attract and/or activate immune effector cells to tumours [76,77].Several ADCs containing cytotoxic radioisotopes, notably the CD20-targeted drugs ibritumomab tiuxetan, 131I-tositumomab, and 131I-rituximab [78], have shown clinical activity against lymphomas.Prostate cancer (trying to target prostatespecific membrane antigen), glioblastoma (directly attacking EGFR), and gastrointestinal tumors (designed to attack carcinoembryonic antigen) are all being studied with similar treatments [79].Antibodies can transport oligonucleotides, allowing for in vivo selective modification of signal transduction pathways [31]. ", "section_name": "Payloads", "section_num": null }, { "section_content": "The synthetic antineoplastic agent auristatin is produced from dolastatin 10, a natural substance [80].Because dolastatin 10 is a nonspecific toxic chemical, it is not used as a cytotoxic warhead in ADCs.In this class of drugs, synthetic analogues including MMAE and MMAF are currently being employed in ADCs as a cytotoxic payload [81].MMAE is an antimitotic drug that works by preventing tubulin polymerization, which causes cell cycle arrest and apoptosis [82]. Maytansinoids are a second significant family of microtubule-disrupting drugs derived from the benzoansamacrolide maytansine.Tubulin polymerization is inhibited by these medications, resulting in mitotic arrest then cell death [83].Maytansinoids perform the same action as Vinca alkaloids.The cytotoxicity of the maytansinoids, on the other hand, was over 100 times that of the Vinca alkaloids [84].Maytansinoids have failed in human trials as anticancer treatment due to a lack of tumour selectivity and substantial systemic toxicity.Maytansinoids' potent cytotoxicity can be used as a targeted delivery vehicle, notably in the form of antibody-maytansinoid conjugates (AMC). ", "section_name": "Microtubule-disrupting agents", "section_num": null }, { "section_content": "Calicheamicins are a kind of enediyne antitumor antibiotic produced from the Micromonospora echinospora bacterium [85].Calicheamicin binds to the minor groove of the TCC TAG GA DNA sequence and prevents it from replicating [86].The payload in the ADC design is N-acetyl-calicheamicin, a calicheamicin derivative [87].Gemtuzumab ozogamicin, sometimes known as Mylotarg, is the name of this ADC.It consists of a humanised IgG4 mAb conjugated to a calicheamicin payload that targets the CD33 surface antigen, which is present in 85-90% of individuals with acute myeloid leukaemia [88]. Duocarmycin is a natural chemical generated from bacterium strains of the Streptomyces genus [89].Duocarmycin is another DNA minor groove-binding alkylating agent.By binding to the minor groove of DNA and causing persistent alkylation of DNA, this family of medicines affects nucleic acid architecture and thus structural integrity [90].Yu and colleagues' work recently highlighted an example of duocarmycin application in ADC setting [91].Promiximab-DUBA, a new ADC against CD56, was described in this work.In this ADC, an anti-CD56 hIgG1 antibody is linked to the payload duocarmycin via a reduced interchain disulfide linker.In vitro and in vivo, this novel ADC showed significant cytotoxic effect against cancer cells.Doxorubicin works by intercalating DNA, which prevents DNA synthesis [92].One well-known example of doxorubicin-based ADC design is the milatuzumab-conjugated doxorubicin ADC (IMMU-1010), which has been employed in phase I/II clinical studies for the treatment of CD74-positive relapsed multiple myelomas [93]. ", "section_name": "DNA-damaging agents", "section_num": null }, { "section_content": "Preclinical studies are conducted to determine a starting dose for human trials as well as to assess the product's toxicity.Antibody/antigen binding studies, in vitro cytotoxicity testing, in vivo anti-tumor efficacy studies, pharmacokinetics, and toxicological studies in rodents and nonhuman primates should all be included in the preclinical evaluation of ADCs [94].ADCs are given intravenously into the circulation to avoid stomach acid and proteolytic enzyme degradation of the mAb [95].The mAb component of ADCs must be expressed selectively on tumor cells and not on normal cells in order to identify and bind to the target antigens [96] (Fig. 3). ADCs combine antibody and cytotoxic drug activities to provide various modes of action and pharmacokinetic properties [18].The complexities of ADC activity in the clinic are only now becoming recognized [97].The antigen-ADC complex is absorbed by receptor-mediated endocytosis (clathrin-or caveolae-mediated endocytosis) or pinocytosis [98,99] (Fig. 3).Internalization causes the cell membrane to bud inward, resulting in the formation of an early endosome, which grows into a late endosome before joining with lysosomes [100].The antigen-ADC complex is then broken down [100].Antimicrotubular medications, for example, are small compounds that can cross the lysosomal membrane and into the cytosol [101].In some cases, the cytotoxic payload may be ejected from the cell or discharged into the tumor microenvironment after being liberated in the cytoplasm.Bystander death occurs when the cytotoxic payload harms cells that do not display the target antigen [102,103].This effect is influenced by a number of factors, including the type of linker employed and the properties of the payload, although the presence of cell-permeable payloads enhances the effect [72].The cell-killing path is determined by the payloads used.Cell death is caused by auristatins and Fig. 3 Mechanism of action of ADC maytansinoids interfering with microtubulins, whereas cell death is caused by calicheamicins and duocarmycins intercalating DNA [7].The ability of an ADC to produce cytotoxicity is determined by a number of factors, including the characteristics of the target antigen's properties, the choice of an antibody, the creation of a stable linker, and the conjugation of effective payloads. Antibodies reach tumour cells by passive diffusion after leakage from capillaries, which results in sluggish and inefficient absorption [104][105][106][107].The Fab and Fc portions of ADCs have an influence on antibodydependent cellular cytotoxicity (ADCC), complementdependent cytotoxicity, and antibody-dependent cellular phagocytosis [108,109].T-DXd and T-DM1, for example, share the same ADCC-competent IgG1 backbone and elicit ADCC in vivo, implying that ADCs could be used as immunotherapy [41,110].Antigen-dependent endocytosis or antigen-independent pinocytosis can be used to internalize ADCs, with clathrin-mediated endocytosis being the most prevalent [111][112][113][114]. Early endosomes are more likely to release payloads with acid-cleavable linkers, whereas late endosomes or lysosomes are more likely to release payloads with enzymatically cleavable linkers [111].Certain ADCs can have a 'bystander effect' on neighboring cells, regardless of the compartment into which the payload is delivered [72]. ", "section_name": "Mechanism of action of ADCs", "section_num": null }, { "section_content": "The failure or ineffectiveness of a treatment is defined as drug resistance.Such failure/reduction may have arisen as a result of drug therapy (secondary or acquired resistance), or it may have existed from the start of treatment (primary or de novo resistance).Resistance to ADCs might theoretically be equivalent to resistance to the ADC's individual components, namely the mAb and the cytotoxic agent.Despite the need for more research, existing clinical data reveal that patients who develop trastuzumab resistance with a taxane react to T-DM1 [115], implying that T-DM1 action is unrelated to previous therapy lines such as anti-HER2 medications or chemotherapies.Because ADCs are targeted therapeutics, fluctuations in the antigen levels detected by the mAb could be a source of resistance.Loganzo and colleagues [116], for example, used many cycles of anti-HER2 trastuzumab-maytansinoid ADC therapy to generate a variety of T-DM1-resistant breast cancer cell lines.Gemtuzumab ozogamicin is consumed by high amounts of CD33 in the blood (GO), [117].For HER2, truncation of the antigen's ectodomain or the masking by components of the extracellular matrix have been proposed as mechanisms of trastuzumab resistance [118].However, masking or shortening of the epitope as mechanisms of ADC resistance have yet to be reported in preclinical animals. A common mechanism of chemotherapeutic resistance is the clearance of the medication from the cellular cytoplasm via ATP-binding cassette (ABC) transporters [119].Because many cytotoxic medicines are ABC transporter substrates, ADC resistance may be caused by these drug efflux pumps [120].In preclinical studies, PgP/MDR1 expression was observed in AML cells that were resistant to GO [121]. One recently reported mechanism of T-DM1 resistance is the drug's action on cyclin B, a cell-cycle protein involved in the G2-M transition.T-DM1 produces a rise in cyclin B in HER2 + breast cancer cells that are sensitive to the treatment, but not in cells that are resistant to the drug 122 .Furthermore, suppressing cyclin B led in drug resistance.T-anticancer DM1's effect coincided with cyclin B buildup in a patient cohort of 18 HER2 + breast cancer fresh explants.These findings have clinical implications since cyclin B induction could be utilized as a biomarker for T-DM1 sensitivity. Activation of downstream signaling pathways can lead to resistance to ADCs.In primary AML cells, GO resistance has been associated to increased PI3K/AKT activation in vitro.In this study, MK-2206, an AKT inhibitor, dramatically sensitized resistant cells to GO or free calicheamicin [123].In a clinical trial, the safety and early evidence of the efficacy of combining T-DM1 and a PI3K inhibitor are being studied (Clinical trials identifier: NCT02038010).ADC sensitivity may be influenced by variations in apoptosis regulation.BAX and BAK, two pro-apoptotic proteins, have previously been linked to the regulation of GO sensitivity in AML [124]. ", "section_name": "Mechanism of resistance against ADC", "section_num": null }, { "section_content": "Resistance to ADCs has been one of the problems limiting these medications' clinical success.ADCs' modular nature allows for the modification of some of its components in order to create novel compounds able to overcome resistance.Increased expression of drug efflux pumps is one of the most common mechanisms of ADC resistance.Changing the cytotoxic agent for medicines or poisons that are poor efflux substrates is one way to get around this.In AML animal models, for instance an anti-CD33 antibody conjugated to PBD, vadastuximab talirine, showed strong effectiveness, even in those where GO had little effect [125]. A second option is to change the linker's hydrophilicity, that can diminish MDR because MDR1 transports hydrophobic chemicals more efficiently than hydrophilic substances.Polar linkers such as sulfo-SPDB (58) and mal-PEG4-N-hydroxysuccinimide have showed enhanced effectiveness against MDR1 + animals [120]. To improve ADCs, the linker-cytotoxic structure can be changed [126].Because tumour heterogeneity is a major problem in cancer, ADCs may be unable to destroy low-antigen-expressing cells. Resistance could also be overcome by new mAb forms, such as bispecific or biparatopic ADCs.In the case of HER2, this has been demonstrated.The first biparatopic ADC, which targeted two nonoverlapping HER2 epitopes, was demonstrated to cause HER2 receptor clustering, which resulted in significant internalization and degradation, as well as anticancer activity in T-DM1-resistant tumor models [50].This biparatopic ADC is now being tested in a number of phase I studies in patients who have failed or are ineligible for HER2targeted therapies. Finally, it appears that combining ADCs with other immunotherapies is a promising strategy [127].The addition of ADCs to immune checkpoint inhibitors may improve treatment response by increasing the recruitment of CD8 + effector T lymphocytes to tumor tissues. ", "section_name": "Resistance-breaking and ADC-based therapy optimization strategies", "section_num": null }, { "section_content": "Several initial tests of ADCs revealed significant adverse effects (AEs) [49].ADCs were created with the primary purpose of increasing tumor targeting and decreasing the toxicities caused by conventional chemotherapy drugs.Surprisingly, cardiac toxicity appears to be rarer with HER2-targeted ADCs than with trastuzumab that has not been conjugated (although the frequency of those AEs still requires an appropriate monitoring).The reason for this apparent difference is unknown; higher cardiotoxicity could be expected if an ADC delivered a cytotoxic payload directly into HER2-expressing cardiomyocytes, but this effect has never been demonstrated in clinical studies.Payload release in the bloodstream, non-malignant tissues, or the TME, as well as the payload's following effects in non-tumour tissues, could be implicated for off-target toxicities [128].The target antigen's expression pattern influences the cytotoxic drug's distribution, which can sometimes result in serious \"ontarget, off-tumor\" toxicities which are not always payload related.In the early 1990s, the ADC BR96-doxorubicin was seen to be highly efficient in mouse xenograft models of a variety of tumor types; however, unlike mice, this antigen is found in non-malignant organs in humans, particularly the gastrointestinal system.T-DXd and trastuzumab duocarmycin, two HER2-targeted ADCs with distinct payloads, both cause pulmonary toxicity through an unknown mechanism [129]. Many ADCs may detect the target antigen in nontumor tissues, but not at sufficient levels to cause damage.Other proteins, such as TROP2, the sacituzumab govitecan target, are expressed in a number of non-malignant organs, but they are only accessible if they are abnormally expressed on the surface of some tumor cells [50,51]. ", "section_name": "ADCs toxicities", "section_num": null }, { "section_content": "Tumors that have been heavily pre-treated have a lot of genetic instability, which causes inter-and intra-tumoral heterogeneity, as well as hypoxic and immunosuppressive TMEs that limit medication penetration [130]. Enfortumab vedotin, a nectin 4-targeted ADC with an MMAE microtubule inhibitor payload, achieved a 44 percent ORR in patients with urothelial metastatic carcinoma previously treated with platinum-based chemotherapy and immune-checkpoint inhibitors [131]. In patients with advanced-stage HER2-positive breast cancer who had received prior T-DM1 and five additional regimens, T-DXd, a next-generation HER2-targeted ADC, achieved an ORR of 60.9 percent (36).T-DM1 is a combination of trastuzumab anti-HER2 antibody and a microtubule-targeting payload (DM1) that has showed promise in patients who have developed resistance to trastuzumab and other microtubule-targeting chemotherapies such as taxanes and vinca alkaloids.T-DXd, which contains a TOPO1 inhibitor, has shown clinical activity (ORR 51%) against gastrointestinal cancers that are only moderately responsive (ORR 14%) to irinotecan, another TOPO1 inhibitor [132,133].As previously stated, intratumoural heterogeneity is a primary cause of targeted therapeutic resistance [134].The bystander effect appears to boost the efficiency of ADCs with cleavable linkers and membrane-permeable payloads, emphasizing chemotherapy's indiscriminate cytotoxicity targeting antigen-negative cells in close proximity to antigen-positive cells [71,135]. There is an obvious need for improved predictive biomarkers to direct ADC therapy [52,136].IHC is the principal approach for measuring the expression of target proteins.IHC, on the other hand, is at best a semi-quantitative assay, and a number of cut-offs have been used to define target positive without explanation.Cytotoxicity may or may not be proportional to targeted antigen expression after a particular density of a certain cell-surface antigen is achieved for ADC activity [6].Meanwhile, some basic concepts can be used to guide the combination of an ADC with a certain cancer type in order to improve treatment success.We anticipate that stable ADCs with non-cleavable linkers will be most beneficial in situations where the target antigen is highly and uniformly overexpressed in a tumor-specific manner [137].This method is likely to effectively eliminate cancer cells while minimising systemic side effects.Labile and/or cleavable ADCs, on the other hand, are expected to rely on the bystander effect to overcome tumour heterogeneity or low-level target expression, often at the expense of off-target effects [71,138].Using irreversible kinase inhibitors against the ADC target concurrently (for example, neratinib with HER2-targeted ADCs) can increase antigen internalization and thus ADC endocytosis and activity [47].ADCs are now being evaluated in over 20 clinical trials in conjunction with approved or investigational immunotherapies.This method is founded on the idea that ADC-mediated cell death and tumor-infiltrating lymphocyte recruitment assist immune effector cells in recognizing immunologically 'cool' tumors and/or improving ADC function. ", "section_name": "Strategies to improve ADCs efficacy in clinics", "section_num": null }, { "section_content": "Of the 110 mAb preparations currently approved by the FDA and/or EMA, 46 (including 13 antibody-drug conjugates) recognise 29 different targets for cancer treatment, and 66 recognise 48 different targets for non-cancer disorders.An updated recent list of FDA approved ADCs against various cancers are provided by recent reviews [18,126].Despite their specific targeting and the expected reduced collateral damage to normal healthy non-involved cells, mAbs can cause type I (anaphylaxis, urticaria), type II (e.g., hemolytic anaemia, possibly early-onset neutropenia), type III (serum sickness, pneumonitis), and type IV (Stevens-Johnson syndrome, toxic epidermal necrolysis) hypersensitivities, as well as other [139].The release of a cascade of cytokines associated with inflammatory and immunological processes is a feature shared by the majority of these syndromes.Antibodies targeting the epidermal growth factor receptor may cause non-immune papulopustular and mucocutaneous eruptions. ", "section_name": "Inflammatory responses against ADCs", "section_num": null }, { "section_content": "ADCs are a novel cancer treatment that combines pharmacogenetic testing with tailored medication.They successfully minimise the systemic toxicity of chemotherapy and provide novel therapeutic alternatives for diseases with poor prognoses and few treatment options.ADCs could be a potential therapy option where specific targeting by antibodies is possible and cell death of the target is the therapeutic goal.ADCs are unique, potent, and unpredictable, and clinical and translational researchers are only beginning to grasp them.Overall, a better understanding of ADC processing and the events that happen after antibody-antigen contact would be tremendously valuable to the ADC development field T. The potential of such a pharmacological platform for cancer treatment could be far-reaching and potentially transformative if the complexity of ADC-tumor interactions can be better understood and utilized.The development of next-generation ADCs with site-specific linker technology, enhanced mAb selectivity, and more effective cytotoxic payloads is presently underway, as are clinical trials to determine the optimum ADC dosage strategies. ", "section_name": "Conclusions", "section_num": null } ]	[ { "section_content": "All images are designed and developed by Biorender. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).The APC will be covered by Mansoura University, Egypt. ", "section_name": "Funding", "section_num": null }, { "section_content": "All data are available in the manuscript. ", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "Author contributions \"H.M. wrote the main manuscript text and prepared Figs.123.AH and CC reviewed the manuscript\".All authors read and approved the final manuscript. Ethics approval and consent to participate Not applicable. This is a single author paper. No competing interests are declared. • fast, convenient online submission • thorough peer review by experienced researchers in your field • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Ready to submit your research Ready to submit your research ?Choose BMC and benefit from: ? Choose BMC and benefit from: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Declarations", "section_num": null }, { "section_content": "Author contributions \"H.M. wrote the main manuscript text and prepared Figs.123.AH and CC reviewed the manuscript\".All authors read and approved the final manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "Ethics approval and consent to participate Not applicable. ", "section_name": "Declarations", "section_num": null }, { "section_content": "This is a single author paper. ", "section_name": "Consent for publication", "section_num": null }, { "section_content": "No competing interests are declared. • fast, convenient online submission • thorough peer review by experienced researchers in your field ", "section_name": "Competing interests", "section_num": null }, { "section_content": "• support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year ", "section_name": "• rapid publication on acceptance", "section_num": null }, { "section_content": "At BMC, research is always in progress. ", "section_name": "•", "section_num": null }, { "section_content": "Ready to submit your research Ready to submit your research ?Choose BMC and benefit from: ? 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10.3390/cancers12030574	Targeting Mitochondrial Apoptosis to Overcome Treatment Resistance in Cancer	<jats:p>Deregulated cellular apoptosis is a hallmark of cancer and chemotherapy resistance. The B-cell lymphoma 2 (BCL-2) protein family members are sentinel molecules that regulate the mitochondrial apoptosis machinery and arbitrate cell fate through a delicate balance between pro- and anti-apoptotic factors. The recognition of the anti-apoptotic BCL2 gene as an oncogenic driver in hematological malignancies has directed attention toward unraveling the biological significance of each of the BCL-2 superfamily members in cancer progression and garnered interest in the targeting of apoptosis in cancer therapy. Accordingly, the approval of venetoclax (ABT-199), a small molecule BCL-2 inhibitor, in patients with chronic lymphocytic leukemia and acute myeloid leukemia has become the proverbial torchbearer for novel candidate drug approaches selectively targeting the BCL-2 superfamily. Despite the inspiring advances in this field, much remains to be learned regarding the optimal therapeutic context for BCL-2 targeting. Functional assays, such as through BH3 profiling, may facilitate prediction of treatment response, development of drug resistance and shed light on rational combinations of BCL-2 inhibitors with other branches of cancer therapy. This review summarizes the pathological roles of the BCL-2 family members in cancer, discusses the current landscape of their targeting in clinical practice, and highlights the potential for future therapeutic inroads in this important area.</jats:p>	[ { "section_content": "tumor cells with the tumor microenvironment adds to chemo-resistance [5].Tumor heterogeneity and the existence of cancer stem cells, may further limit treatment response. Increasingly, dysregulation of drug-induced autophagy and apoptosis has been recognized as a key mechanism of carcinogenesis and chemotherapy resistance, whereby the surviving cancer cell continues to accumulate oncogenic mutations which further propagate tumor progression [6].Targeting apoptosis therefore holds promise in overcoming resistance to cancer therapy.Recently, venetoclax (ABT-199) has successfully achieved USA Federal Drug Administration (FDA) approval for the treatment of patients with chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), confirming that apoptosis-targeting strategies have finally come of age.In this article, we discuss the roles of the BCL-2 superfamily in carcinogenesis and treatment resistance, and review the successes and failures of strategies targeting the BCL-2 family members in cancer therapy. ", "section_name": "", "section_num": "" }, { "section_content": "", "section_name": "The BCL-2 Superfamily and Its Role in Apoptosis", "section_num": "2." }, { "section_content": "Apoptosis is effected via the intrinsic and extrinsic pathways.The extrinsic, or death-receptor mediated pathway, is initiated when cell death receptors such as Fas, TNFR1, TRAIL-R1, TRAIL-R2, DR3 and DR6, interact with their ligands on the cell surface.Activation of Fas, TRAIL-R1 or TRAIL-R2 leads to the formation of a \"death-inducing signaling complex\" (DISC) and triggers a cascade of caspase activation culminating in apoptosis, while the activation of other receptors induces apoptosis by triggering different signaling pathways such as NF-κB [7,8].Detailed discussion of the extrinsic pathway and its targeting is beyond the scope of this review.The intrinsic, or mitochondrial pathway, responds to intracellular apoptotic stimuli such as viral infection, oxidative stress, calcium flux and DNA damage caused by drug or radiation exposure [9,10].When committed to apoptosis, mitochondria outer membrane permeabilization (MOMP) is the decisive event through which cytochrome c and the second mitochondria-derived activator of caspase (SMAC) are released into the cytoplasm, triggering apoptosome assembly and caspase 9 activation [7].Downstream executioner caspases 3, 6 and 7 cause cellular dismantlement and cytoskeletal protein degradation, which lead to the classic morphological features of crenation, DNA condensation and, ultimately, cell death [11]. The BCL-2 family members are central regulatory players in the intrinsic mitochondrial apoptotic program, and their interplay controls cell fate [12] (Figure 1).More than 25 BCL-2 family members have been identified.Advances in structural resolution of these members have categorized them into three subfamilies-(1) the multidomain anti-apoptotic members (BCL-2, BCL-xL, MCL-1, BCL-w, BCL-B/Boo and BFL-1/A1), (2) the multidomain pro-apoptotic members (BAX, BAK), and (3) the BH3-only members (BAD, BID, NOXA, HRK, BMF, PUMA, BIM).Multi-domain members in (1) and ( 2) have four BCL-2 homology (BH) domains (BH1, BH2, BH3 and BH4) each, while the BH3-only members are comprised of only a single short BH3 domain [13][14][15].The BH3 motif is composed of 9 to 15 amino acids and is uniquely conserved across all BCL-2 family members [16].BH3 interactions are responsible for orchestrating the BCL-2 interactome via a BH3-into-hydrophobic groove mechanism [17,18], which allows the formation of homo-and heterodimers that control apoptotic function [19].Minor alterations in the amino acid sequences of the binding grooves and BH3 domains control the specificity of these interactions. The balance between pro-and anti-apoptotic family members determines if intrinsic apoptosis will proceed.When pro-apoptotic BAX or BAK are liberated, they are able to oligodimerize in the outer mitochondrial membrane, leading to the formation of a mitochondrial membrane pore which commits the cell to MOMP.The BH3-only proteins have complex roles as death sentinels that link apoptotic signals to the intrinsic pathway, and are divided between two roles-either as \"direct activators\" (tBID, BIM, PUMA) of BAX and BAK, exposing the BH3 domain of BAX and BAK to facilitate oligodimerization [20]; or as \"inactivators\" or \"sensitizers\" (BAD, BIK, BMF, HRK, NOXA) by binding with anti-apoptotic BCL-2 thus allowing BAX/BAK to be unrestrained to trigger MOMP [21] (Figure 1).The intrinsic apoptotic pathway and interactions between pro-and anti-apoptotic B-cell lymphoma 2 (BCL-2) family members.Intrinsic pathway apoptotic stimuli such as viral infection, oxidative stress, calcium flux and DNA damage lead to changes in the balance of pro-and anti-apoptotic BCL-2 family members.The anti-apoptotic proteins act to prevent BAX/BAK activation.Activator BH3-only proteins (PUMA, tBID, BIM) inhibit all anti-apoptotic members, whereas sensitizer BH3-only proteins interact and engage selective anti-apoptotic members, allowing BAX/BAK oligomerization and indirect activation.Oligomerization of BAX/BAK in the mitochondrial membrane commits the cell to mitochondria outer membrane permeabilization (MOMP), and triggers a downstream caspase cascade which ends in apoptosis. ", "section_name": "The BCL-2 Superfamily Controls the Intrinsic Apoptosis Pathway", "section_num": "2.1." }, { "section_content": "The importance of apoptosis in homeostasis requires that it is tightly regulated.The canonical roles of the BCL-2 subfamilies suggest that apoptosis may be triggered through inactivation of the anti-apoptotic multidomain subfamily proteins, or an increase in concentration of BH3-only proteins.Yet the BCL-2 puzzle has proven far more complex and often unpredictable, contributed by pleiotropic effects of multiple signaling controls, as well as post-transcriptional and post-translational modification processes, in modifying the affinity between BCL-2 family members [16].For example, PI3K-Akt activation leads to phosphorylation and inactivation of BAD [22], leaving BCL-2 free to inhibit apoptosis, while increasing the expression of anti-apoptotic genes [11].Similarly, activation of the extracellular signal regulated kinase (ERK) pathway results in increased transcription of the anti-apoptotic subfamily of BCL-2 members, and increased ubiquitination and subsequent degradation of pro-apoptotic members, leading to cell survival [23].ERK-mediated phosphorylation of MCL-1 at T163 was further shown to stabilize MCL-1, leading to suppression of apoptosis in various hematological malignancy cell lines [24]. In the nucleus, genomic alterations such as chromosomal translocations and gene amplifications may increase BCL-2 levels.A notable example would be in CLL, where deletion of chromosome 13q in >50% of patients leads to silencing of the microRNAs miR-15 and miR-16, which are responsible for degrading BCL-2 RNA, resulting in BCL-2 overexpression [25].Post-translational modifications moderate protein functions through ubiquitination, proteolysis, phosphorylation and proteasomal degradation [26].Phosphorylation of BCL-2 at S70 [27] has been described to alter its anti-apoptotic ability [28] and confer resistance to taxane chemotherapy [29].Specific BIM phosphorylation sites have the ability to affect its BCL-2 binding capability, resulting in an anti-apoptotic phenotype, while mutations at other phosphorylation sites (Ser-55, -65 and -73) tag BIM for proteasomal degradation, increasing therapy resistance [30].Phosphorylation of BAX at specific residues (S184), mediated by Akt activation, has been suggested to switch BAX from pro-to anti-apoptotic in phenotype, by allowing it to sequester activator BH3 proteins [18]. ", "section_name": "Regulation of BCL-2 Family Members", "section_num": "2.2." }, { "section_content": "Dysregulation of the anti-apoptotic members BCL-2, BCL-xL and MCL-1 have been widely described in carcinogenesis, cancer progression and chemotherapy resistance [31].Cancer cells often upregulate anti-apoptotic BCL-2 proteins, thus tilting the ratio of anti-versus pro-apoptotic members to fall in favor of apoptosis evasion, even in the presence of stimuli from chemotherapeutic agents [6].CLL is considered one of the classical hematological malignancies attributable to failure of apoptosis.Nearly all CLL patients have increased BCL-2 expression.Repression of BCL-2 at the post-transcriptional level allowed for the induction of apoptosis in CLL cell lines [25].BCL-2 overexpression is a key event in follicular lymphoma (FL), driven by pathological chromosomal t(14; 18) translocation, whereby the BCL2 oncogene is pathogenically translocated to the immunoglobulin heavy chain (IGHV) gene locus, leading to its amplification.In diffuse large B-cell lymphoma (DLBCL), concomitant overexpression of BCL-2 and MYC is classified as a \"double-hit\" DLBCL, which is associated with a dismal prognosis, high risk for relapse, resistance to standard chemotherapy and justifies upfront escalation to more intensive treatment.These observations have fueled strategies therapeutically targeting the anti-apoptotic BCL-2 members in cancer treatment. An interesting and somewhat non-canonical aspect of the functional biology of BCL-2 is ability to maintain a mild mitochondrial pro-oxidant milieu while preventing deleterious levels of reactive oxygen species (ROS) production triggered by oxidative stressors through the regulation of cytochrome c oxidase activity [32].This mechanism appears to be the result of an interaction between BCL-2 and the subunit COX Va that shifts the ratio of COX Va to COX Vb subunits, thus modulating cytochrome c oxidase activity.The modulation of ROS production by BCL-2 expression is a critical component of its anti-apoptotic activity as cells subjected to oxidative stress inducers modulate their mitochondrial redox metabolism to buffer the excess ROS production, thereby promoting cell survival [33].In addition, the pro-oxidant milieu generated through superoxide anion production by an increased expression of BCL-2 was shown to be linked to an interaction between BCL-2 and the small GTPase Rac1, a critical regulator of NADPH oxidase, responsible for superoxide production [34].Interestingly, a mild to moderate increase in intracellular superoxide anion (pro-oxidant state) has also been shown to impact the phosphorylation status of BCL-2, specifically at S70 via the generation of peroxynitrite (a reaction product of superoxide and nitric oxide).This involves peroxynitrite mediated nitrative modification of the regulatory subunit B56δ of the protein phosphatase 2A (PP2A), which prevents holoenzyme assembly and results in the sustained S70 phosphorylation of BCL-2 to stabilize its anti-apoptotic activity [35].These findings provide evidence for an intricate crosstalk between BCL-2 and cellular redox metabolism, thereby delineating a novel facet in the biology of this death regulatory protein with potential therapeutic implications. MCL1 is one of the most highly amplified genes in human cancers [36].In hematological malignancies, increased levels of MCL-1 have been described in multiple myeloma (MM) [37], DLBCL [38], AML, chronic myeloid leukemia (CML) and mantle cell lymphoma (MCL).Many chemotherapeutic agents affect apoptosis through the reduction of MCL-1 levels.In CLL cell lines, up-regulation of MCL-1 after co-culture with stroma was linked to fludarabine resistance [39].Conversely, knock-down of MCL1 in mice models not only triggered apoptosis of transformed AML cells but also salvaged AML-afflicted mice from disease progression [40]. Finally, elevated BCL-xL expression has also been observed in MM [41] and non-Hodgkin's lymphoma (NHL), and is implicated in their progression.In one study, transgenic mice with overexpression of BCL-xL readily developed lymphomas [42].This is further supported by studies showing that interactions between pro-apoptotic BCL-xL and anti-apoptotic BIM control the apoptosis rate in MYC-related lymphoma [43]. Conversely, the loss of pro-apoptotic proteins appears to be relatively uncommon.Somatic inactivation of BAX (and BAK) has been reported in both solid and hematological cancers [44].Deletion or silencing of NOXA, PUMA or BIM expression has been described in the pathogenesis of hematologic cancers and their response to chemotherapy [45,46].Of note, BIM is deleted in 17% of MCL [47], while BAX mutations occur in 20% of hematologic cancers such as CLL, FL, MCL and NHL.In mouse fibroblast models, loss of both BAX and BAK led to resistance to chemotherapy-induced apoptosis [48].Additionally, loss of BAX in colon cancer cells led to 5-fluorouracil resistance [49]. Indeed, the complex roles of the BCL-2 family members have created immense potential for targeting.Progressive and stepwise improvements in our mechanistic understanding of apoptosis have since allowed for the identification of entry points into this network, toward the promise of optimal therapeutic targeting in cancer.In the next section, we discuss the historical advancements in BCL-2 family targeting that have led to the success of venetoclax in modern day hematological malignancy treatment, and delve into upcoming novel strategies. ", "section_name": "Dysregulation of BCL-2 Family Members in Carcinogenesis and Treatment Resistance", "section_num": "2.3." }, { "section_content": "", "section_name": "Targeting the BCL-2 Superfamily: A Summary of the Current Landscape", "section_num": "3." }, { "section_content": "ASOs were the first approaches employed for BCL-2 inhibition.These are complementary strands that hybridize with and silence anti-apoptotic BCL-2 subfamily mRNA, leading to hydrolysis of the mRNA and promoting apoptosis [50,51].Oblimersen is an 18-antisense oligonucleotide complementary to the first six codons of BCL-2 mRNA that was evaluated in a variety of hematological malignancies.Promising response rates were seen when combined with standard chemo-immunotherapy [52,53], and also allowed lower doses of chemotherapy to be administered.Reduced BCL-2 mRNA and protein levels were noted in AML patients who achieved a complete response (CR) with oblimersen, providing proof-of-principle of its mechanism of action [53].Common toxicities included fever, fatigue, gastrointestinal side effects and night sweats.However, on several phase III studies, no survival advantage could be shown for oblimersen addition [52].Despite this, several patients treated with oblimersen on study appeared to derive durable benefit from this drug [52].Other ASOs under evaluation include SPC2996, PNT2258 and bispecific ASOs targeting BCL-2/BCL-xL. ", "section_name": "Antisense Oligonucleotides (ASO)", "section_num": "3.1." }, { "section_content": "The recognition of BH3-only proteins as natural inhibitors of BCL-2 proteins led to the development of BH3-mimetics.These small molecules are homologous to the BH3 domains of anti-apoptotic BH3-only proteins, and bind competitively to the hydrophobic groove of anti-apoptotic proteins, displacing BAX/BAK or pro-apoptotic BH3-only molecules, inducing apoptosis.Venetoclax (ABT-199, Abbvie Inc, North Chicago, IL, USA), which targets BCL-2, was the front-runner inhibitor in developmental pipelines for BH3-mimetics, and its FDA approval across four indications represents a major milestone in this field.To date, BH3-mimetics specifically inhibiting BCL-2, MCL-1 and BCL-xL, respectively are undergoing evaluation. ", "section_name": "BH3-Mimetics", "section_num": "3.2." }, { "section_content": "Gossypol acts as a pan-BCL-2 family inhibitor [54], and has both BAX/BAK-dependent [55] and -independent [56] mechanisms of action.In preclinical studies, gossypol demonstrated promising activity through activation of the intrinsic apoptotic pathway in CML [57], NHL [58] and MM [59].In-vivo studies in mouse models [60] showed significant slowing of tumor growth when gossypol was combined with CHOP chemotherapy, compared to either CHOP or gossypol alone [60].However, gossypol has significant off-target side effects including dose-limiting thrombocytopenia, preventing it from advancing into clinical trials.The more potent, and orally-available enantiomer of gossypol, AT-101 progressed further in its development.However, despite promising preclinical data, early phase studies of AT-101 in combination with docetaxel for prostate cancer or non-small cell lung cancer (NSCLC) did not show improved outcomes [61,62] (Table 1).In CLL, AT-101 with rituximab showed only modest efficacy [63].Gastrointestinal toxicities (such as nausea, vomiting and ileus), fatigue and neutropenia were the most common side effects noted [63].Newer analogues of AT-101 include TW37 and TM-1206, which have improved affinity to BCL-2, MCL-1 and BCL-xL. ", "section_name": "Gossypol and AT-101", "section_num": "3.2.1." }, { "section_content": "Obatoclax (GX15-070; Teva Pharmaceutical Industries Ltd, Parsippany, NJ, USA) is a relatively weak polypyrrole pan-BCL-2 family inhibitor that is able to bind to anti-apoptotic BCL-2, BCL-xL, BCL-w, BCL-B, BFL-1/A1 and also MCL-1 with sub-micromolar affinity, allowing BAK/BAX oligomerization and cell death [64].Obatoclax is also purported to have BCL-2 independent mechanisms, via its effect on the Akt/mTOR signaling pathway [65], which increases the possibility of off-target toxicity.In various hematological malignancy cell lines, and in-vivo mouse models, obatoclax monotherapy showed anti-cancer activity [65,66].However, raising obatoclax serum levels to clinically effective concentrations in mice models was associated with severe neurotoxicity.Accordingly, when obatoclax was tested in phase I/II trials for AML, CLL, acute lymphoblastic leukemia (ALL), myelodysplastic syndrome, MCL and classical Hodgkin lymphoma (HL) [67][68][69][70], only limited clinical activity was observed (Table 1).Common adverse events included mood disturbances and gastrointestinal side effects, while high grade toxicities appeared to be mainly hematological [68].Development of obatoclax has been discontinued. ", "section_name": "Obatoclax", "section_num": "3.2.2." }, { "section_content": "ABT-737 (Abbvie Inc, North Chicago, IL, USA) was the \"first-in-class\" small molecule inhibitor designed as a BH3 mimetic of BAD.ABT-737 was shown to bind with a much higher affinity (sub-nanomolar concentrations) and more selectively, compared to obatoclax, to BCL-2, BCL-xL and BCL-w [71].Activity of ABT-737 was shown in MM, and AML cell lines (Table 1).Notably, in CML cell lines, ABT-737 plus imatinib reduced the development of BCL-2 driven imatinib-resistance [72].The specific binding of ABT-737 to its intended targets resulted in an increase in MCL-1 expression and phosphorylation thus bypassing the effect of ABT-737 and leading to ABT-737-resistance in AML cells [73].Compounding this, the unfavorable pharmacokinetic profile of ABT-737 further spurred the development of newer generations of BH3 mimetics. ", "section_name": "ABT-737", "section_num": "3.2.3." }, { "section_content": "Navitoclax (ABT-263; Abbvie Inc, North Chicago, IL, USA) is a second-generation, orally bioavailable BH3-mimetic.Navitoclax binds preferentially to BCL-2, BCL-xL and BCL-w with nanomolar affinity, specifically disrupting BCL-2 and BCL-xL interactions with pro-death BH3 members.However, navitoclax still lacks the ability to antagonize MCL-1 and BFL-1/A1 dependent interactions.In-vivo xenograft models of small cell lung cancer (SCLC), ALL, NHL, MCL and MM showed promising tumor regression [74].In a phase I study of 29 patients with relapsed or refractory CLL, navitoclax as a single-agent showed an overall response rate (ORR) of 35% in patients receiving a daily dose of at least 110mg, although no CR was observed [75].Despite this, durable responses >12 months occurred, even in patients with the poor prognostic marker, deletion 17p.Furthermore, a higher ratio of BIM to MCL-1 and BIM to BCL-2 correlated with improved efficacy of navitoclax [75].As expected, patients with lower pre-treatment MCL-1 levels had improved response to navitoclax.Prominent thrombocytopenia occurred early after treatment-initiation, this was often dose-limiting [75], and consistent with previous data showing that the homeostasis of mature platelets is dependent on BCL-xL.Currently, navitoclax is increasingly under investigation in solid tumors due to the inherent risks of severe thrombocytopenia in patients with hematological malignancies who are already myelosuppressed (Table 1). Table 1.Early-generation BH3-mimetics inhibiting BCL-2.R/R: relapsed/refractory; CLL: chronic lymphocytic leukemia; HL: hodgkin's lymphoma; MDS: myelodysplastic syndrome; ORR: objective response rate; FL: follicular lymphoma; FR: fludarabine plus rituximab; iwCLL: international workshop on CLL; MCL: mantle cell lymphoma; CR: complete response; PR: partial response; PFS: progression-free survival; RP2D: recommended phase 2 dose; ALL: acute lymphoblastic leukemia; SCLC: small cell lung cancer; NSCLC: non-small cell lung cancer; OS: overall survival.Hydrogen bonds between venetoclax and Asp103 on BCL-2 result in the increased selectivity of venetoclax for BCL-2 compared to previous compounds [85].Venetoclax is an orally-available, extremely potent and selective BCL-2 only inhibitor, and is platelet-sparing [85].Due to its improved therapeutic window, this drug emerged as the front-runner BH3-mimetic, particularly in malignancies which are BCL-2 dependent. ", "section_name": "Navitoclax", "section_num": "3.2.4." }, { "section_content": "As described earlier, the central role of the BCL2 super family in CLL has made this disease a key substrate for studying and developing BCL-2-targeted therapy.In a phase I dose-escalation study of 116 relapsed/refractory CLL and NHL patients treated with venetoclax, ORR of 79% and CR rate of 20% was seen in patients with CLL.This was particularly impressive as the target-population had included heavily-pretreated CLL patients and 90% of patients harbored at least 2 poor prognostic markers, such as chromosome 17p-deletion, 11q deletion, fludarabine-resistance, bulky lymphadenopathy and lack of mutation in IGHV.This potent and rapid cell kill was further confirmed by the unexpectedly high rate of tumor lysis (TLS) in 18% of patients leading to fatalities.Amending the dose schedule to feature a risk-mitigating ramp-up dose, together with monitoring and adequate TLS prophylaxis, helped to prevent this feared side effect.Despite rampant expression of BCL-2 in healthy tissues, other adverse events (AEs) were manageable, such as diarrhea, nausea and neutropenia.Treatment with venetoclax in the dose-escalation cohort resulted in an estimated 2-year overall survival (OS) rate of 84% [86] (Table 2). These impressive results led to a pivotal phase II study of 107 patients with relapsed/refractory deletion-17p CLL treated with venetoclax.ORR with venetoclax was 79%, including CR 8%, and responses were seen regardless of the presence of poor-prognostic markers [86] (Table 2).Specifically in CLL patients treated with venetoclax after progressing on the B cell receptor inhibitors (BCRis) ibrutinib or idelalisib, the phase II M14-032 study reported ORR 67% and time-to-response of 2.5 months.Even in a small exploratory subgroup of 28 patients who had previously received more than 1 previous BCRi, encouraging activity was noted [87] (Table 2). Venetoclax therapy is made even more convincing by its ability to result in unprecedented phenomenon of undetectable minimal residual disease (uMRD), which is defined when there is <1 CLL cell per 10,000 lymphocytes in marrow or peripheral blood.Low or uMRD has been shown to correlate with improvements in OS [88].In a pooled analysis of 2 phase II studies of relapsed/refractory CLL patients treated with venetoclax, the PFS rate was 92.8% in patients achieving uMRD at 24 months on treatment [89,90].The first approval for venetoclax in patients with CLL came in 2016, where patients with 17p deletion were approved to receive venetoclax in the subsequent-line setting.This was later extended to patients with CLL or small lymphocytic lymphoma (SLL) in June 2018, regardless of 17p deletion, in the subsequent-line setting. The combination of rituximab, an anti-CD20 antibody, to venetoclax has also shown to be highly effective and able to achieve high uMRD rates in relapsed/refractory CLL.Preclinical data showed that this combination was able to counteract micro-environmental signals that were contributing to venetoclax resistance in CLL [91].The phase III MURANO study [92] compared venetoclax-rituximab for 6 cycles followed by a 2 year-maintenance treatment, to 6 cycles of bendamustine-rituximab, and showed remarkable improvements in 2-year progression-free survival of 84.9% versus 36.3% [92], as well as 3-year uMRD rate (62% versus 13%) [93].Another phase III study recently reported results comparing venetoclax-obinutuzumab versus chlorambucil-obinutuzumab in previously untreated CLL patients.Venetoclax-obinutuzumab was associated with significantly improved PFS at 24 months (24-month PFS rate 88.2% versus 64.1%), and this benefit was extended to patients with poor prognostic factors [94].These impressive results relating to uMRD, together with pooled analysis data suggesting that venetoclax should be sequenced earlier in treatment paradigms, ultimately led to the FDA indication being expanded to all adult patients with CLL or SLL in May 2019. Venetoclax monotherapy is modestly active in relapsed/refractory AML.Of note, patients harboring IDH1/2 mutations appeared to perform better with venetoclax therapy, with CR rate of 33% [95,96]. Further phase Ib studies have also combined venetoclax with hypomethylating agents based on preclinical models demonstrating synergy [97].When combined with low dose cytarabine, decitabine or azacytidine in untreated elderly patients, CR/CRi rates ranged between 54-68% across studies with a median time to response of 1.2-1.4months, with tolerable toxicity [98,99] (Table 2).This led to a further FDA breakthrough status in November 2018 for venetoclax in combination with hypomethylating drugs for newly-diagnosed elderly AML patients ineligible for intensive chemotherapy. Venetoclax has also shown promising activity in relapsed/refractory MCL.In a phase I trial of 106 patients with relapsed/refractory NHL, patients with MCL had particularly high response rates (ORR of 75%, CR 21% [100].Venetoclax plus ibrutinib was evaluated on a phase II study, which recruited a majority of relapsed/refractory MCL patients, again showing high response rates of ORR 71%, CR 63% [101], and this is being explored further on a phase III study (Table 2).Venetoclax monotherapy appears to be less active in other NHL, in particular relapsed/refractory DLBCL, where only modest response rates of around 18% were noted [100].Similarly, in relapsed/refractory MM, ORR for venetoclax monotherapy was 21% [102].In cell lines, the t(11; 14) (q13;q32) translocation was shown to increase BCL-2:MCL-1 ratio and lead to lower BCL-xL levels, and patients harboring this translocation may benefit the most [103]. As alluded to, venetoclax has heralded the way for the development of other BCL-2 inhibitors.Newer BCL-2 inhibitors in the pipeline include S55746 (Servier, Suresnes, France) which has dual BCL-2/ BCL-xL inhibiting capabilities [104,105] (Table 2).It is likely that the role for BCL-2 inhibitors is likely to expand in cancer therapy, and further results are awaited. ", "section_name": "Drug", "section_num": null }, { "section_content": "BCL-xL dependency has been described across tumor types, aggregating mainly in solid tumors [106].This makes selective BCL-xL inhibition an attractive target, especially in the treatment of venetoclax-resistant cancers.As described earlier, BCL-xL expression in AML, MM and some solid tumor models, is associated with chemotherapy and venetoclax resistance [107,108].WEHI-539 (The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) was the first selective BCL-xL inhibitor published.When bound to BCL-xL, WEHI-539 induced BAK-mediated cell death in SCLC cell lines.When used in osteosarcoma cells that overexpressed BCL-xL, WEHI-539 was able to potentiate the effect of low-dose doxorubicin [109].However, further development of this compound has been halted due to in-vivo toxicity.On-target toxicities of such inhibitors include thrombocytopenia, which occurs rapidly and reversibly, similar to what was observed with navitoclax.Further BCL-xL selective inhibitors under pre-clinical evaluation include A-1155463 and A-1331852 (Abbvie Inc., North Chicago, IL, USA) (Table 3). ", "section_name": "BCL-xL-Selective BH3-Mimetics", "section_num": "3.2.6." }, { "section_content": "In healthy tissues, MCL-1 regulates neural and cardiac cell survival.In cancer, not only has the MCL-1 protein been shown to regulate cell survival in myeloid and lymphoid cancers including MM, AML and NHL [36,110], but MCL1 amplifications have been found in >10% of solid tumor cancer types [106].In triple-negative breast cancer, MCL1 amplification correlates with poor prognosis [111]. Drug development in MCL-1 inhibition is ongoing with several candidate compounds in early phase testing (Table 3).A-1210477 (Abbvie Inc., North Chicago, IL, USA) was the first inhibitor able to disrupt MCL-1-NOXA and MCL-1-BIM2A interactions selectively [107].When used in MM and NSCLC cell lines that showed MCL-1 dependency, A-1210477 triggered MOMP and apoptosis [107].A more potent MCL-1 inhibitor, S63845 (Servier, Suresnes, France) [112], is also undergoing evaluation, and has been shown to have sub-molar affinity to the MCL-1 BH3 binding groove with BCL-2/ BCL-xL binding.In vitro, S63485 induced BAX/BAK-mediated apoptosis in solid tumors, as well as elicited intriguing synergism with tyrosine kinase inhibitors(TKIs) [112].Several other MCL-1 inhibitors [AZD5991(AstraZeneca), AMG-176, AMG-397(Amgen), S64315/MIK665(Novartis)] are currently undergoing phase I clinical trials in a variety of hematological malignancies (Table 3).Despite these advancements, the concern for the development of side effects of MCL-1-targeting agents on cardiac and neurological systems may pose challenges to clinical development of these agents, and further results are awaited. ", "section_name": "MCL-1 Antagonists", "section_num": "3.2.7." }, { "section_content": "Similar to BH3, the BH4 domain is conserved amongst the members of the BCL-2 superfamily.Aside from its crucial role in the anti-apoptotic activity of BCL-2, the BH4 domain also is required for other non-canonical functions of the BCL-2 superfamily, such as in calcium homeostasis at the ER [113].Notably, losing the BH4 domain greatly diminishes the anti-apoptotic function of BCL-2 [114].Targeting BH4 is therefore emerging as a novel strategy in cancer therapy (Table 3). ", "section_name": "Targeting the BH4 Domain", "section_num": "3.3." }, { "section_content": "Interference technologies at the DNA and RNA level utilize a nucleic acid-based approach to block transcription and translation of BCL2 respectively.Silencing BCL2 by utilizing RNA interference (RNAi) technology is still in its infancy.Early data regarding the efficacy of this approach have been generated using ALL cell lines and xenografts [115].PNT2258 (ProNAi Therapeutics Inc., Vancouver, Canada) a first-in-class DNAi drug that consists of a 24-base sequence complementary to regions of DNA that are upstream from sites of gene transcription, thus preventing BCL-2 transcription [116].In pre-clinical studies, PNT2258 was active in BCL-2 driven xenografts, including in NHL, prostate cancer and melanoma [117].Differential activity was seen in different NHL cell lines according to their levels of BCL-2 overexpression.Initial phase I studies of PNT2258 confirmed a safe toxicity profile with tolerable lympho-and thrombocytopenia [118].Initial interesting responses, especially in DLBCL patients, were noted on a phase I study of PNT2258 in relapsed/refractory NHL.However, these results were not corroborated in a phase II study of relapsed/refractory DLBCL, and the development of PNT2258 was subsequently discontinued [119] (Table 3). ", "section_name": "Interference Technology", "section_num": "3.4." }, { "section_content": "", "section_name": "Navigating Anti-Apoptotic BCL-2 Dependency to Tackle Therapy Resistance", "section_num": "4." }, { "section_content": "Despite the excellent results obtained from the use of venetoclax, many patients progress after a period of treatment (acquired resistance), while others do not respond at all (intrinsic resistance).Although BCL-2 is overexpressed in a multitude of solid tumors and hematological malignancies, significant responses to venetoclax monotherapy are limited to only a handful of cancer types.This is because, although BCL-2 may be over-expressed, this may not be reflective of its pathological function. ", "section_name": "BCL2 Dependency in Intrinsic and Acquired Therapy Resistance", "section_num": "4.1." }, { "section_content": "Acquired resistance is also linked to the concept of BCL-2 dependency.Functional redundancy within the BCL-2 superfamily allows acquired resistance to develop by switching reliance on other anti-apoptotic members as a result of treatment pressure.The exact mechanisms of these dynamic inhibitory responses have yet to be defined.In CLL cells, compensatory BCL-xL and BFL-1/A1 upregulation was associated with acquired venetoclax resistance [120,121].In NHL cell lines, prolonged venetoclax treatment also resulted in increased BCL-xL and MCL-1 expression, mediated by Akt signaling [122].Similarly, in lymphoma experiments, resistance to ABT-737 was affected through a shift in BCL-2 family member dependency by the upregulation of MCL-1 or BFL-1/A1 [123].Aberrant NF-kB signaling has been shown to affect resistance to ibrutinib plus venetoclax combination therapy in CLL cells by increasing MCL-1 and BCL-xL expression [124].Venetoclax plus decitabine or azacitidine, CR/CRi: 67%.In patients ≥75 years old or with poor-risk cytogenetics, CR/CRi was 65% and 60% respectively.Median OS was 17.5 months. Treatment was well tolerated. [ ", "section_name": "Concurrent or dominant expression of MCL-1 or BCL-xL may indicate dependency on these family members instead, leading to intrinsic resistance to and limited utility of venetoclax monotherapy.", "section_num": null }, { "section_content": "BH3 profiling is a functional assay technique that has helped to provide clarity on BCL-2 dependency.This technology is able to determine (1) the degree of 'mitochondrial priming' of a cell [138], and (2) BCL-2 member dependency, and hence predict response and resistance to therapies targeting the BCL-2 family [139]. Briefly, an array of functionally-distinct BH3-only proteins are added to isolated mitochondria or permeabilized cells taken from a fresh cancer sample, and allowed to interact with other BCL-2 superfamily proteins at the mitochondrial surface, inducing MOMP.MOMP is measured indirectly by the amount of cytochrome release or by the loss of inner mitochondrial membrane potential (MMP).In cells that have highly-primed mitochondria, BH3-only proteins rapidly induce MMP loss, compared to cells that have low priming.Importantly, the differing specificity of some BH3-only proteins for anti-apoptotic members e.g.: NOXA for MCL-1 will allow the BCL-2 dependency of the cell to be inferred by the degree of MOMP triggered when different BH3-only proteins are added [140]. MM and AML cells with dominant MCL-1 dependency or heterogenous dependency on multiple members-BCL-2, MCL-1, BCL-xL-were predicted to be resistant to BCL-2 inhibitor monotherapy, unless the other members are able to be abrogated [76].In MM and NSCLC, the presence of MCL-1/BAK complexes predicted sensitivity to MCL-1 inhibition with A-1210477 [107].The level of mitochondrial priming inferred from BH3 profiling also provides information regarding the depth of response.In CLL cells, high mitochondrial priming pre-treatment was associated with deeper venetoclax responses [141].In AML cell lines, low mitochondrial priming has been correlated with chemotherapy resistance [142]. New developments in BH3 profiling include whole cell JC-1 based technology that allows easier measurements of cytochrome c release through the use of JC-1, a fluorescent probe.FACS technology now allows BH3 profiling to be performed in polyclonal cell populations [120], potentially providing insight into BCL-2 dependency despite tumor heterogeneity.Furthermore, given the described compensatory upregulation of other anti-apoptotic BCL-2 family members such as MCL-1 and BFL-1/A1 in response to ABT-737 treatment in lymphoma [123] and BCL-xL in response to venetoclax in CLL, identification of these changes via BH3 profiling may allow for sequential use of novel inhibitors such as those against MCL-1 or BCL-xL to combat acquired resistance.Therefore, systematic sequential BH3 profiling has promise as a dynamic biomarker to allow us to document changes in a tumor's anti-apoptotic BCL-2 dependency longitudinally, predict depth of response, and even select the right therapeutic strategy to target specific molecular vulnerability in a personalized approach. ", "section_name": "BH3 Profiling to Define BCL2 Dependency", "section_num": "4.2." }, { "section_content": "", "section_name": "Expanding Clinical Contexts for BCL-2 Targeting", "section_num": "5." }, { "section_content": "Despite the gains we have made through venetoclax in specific clinical contexts, rational combinations of BCL2-targeting therapy with chemotherapeutics and other targeted therapy hold promise to advance treatment paradigms.Venetoclax is currently being combined with different branches of cancer therapy in different hematological and solid malignancies, chosen based on known pathways that are aberrant in specific tumor types (Table 2).Aside from combination with chemotherapy, other important combinations under investigation include those with proteasome inhibitors (bortezomib, carfilzomib), PI3K inhibitors (idelalisib), BTK inhibitors (ibrutinib), CDK inhibitors (dinaciclib, palbociclib), MEK inhibitors (cobimetinib), MDM2 inhibitors (idasanutlin) and other novel agents (Table 2).Co-targeting of different BCL-2 family members to overcome resistance, such as concurrent BCL-2 and MCL-1 targeting or BCL-2/ BCL-xL targeting is also under study (Table 2). The rationale to combine BCL-2 targeted drugs with chemotherapy is based on the understanding of mitochondrial priming.Treatment with BH3-mimetics is expected to raise the mitochondrial priming state, thereby allowing them to act as \"chemosensitizers\" for synergism with cytotoxic chemotherapy [143] (Table 2).Furthermore, this approach holds benefit not just in enhancing cell kill, but also may reduce treatment doses, thus reducing toxic side effects. Venetoclax in combination with BTK inhibitors in CLL and MCL treatment is actively being explored.Samples taken from CLL patients receiving ibrutinib were analyzed in-vitro with the addition of venetoclax, and proved synergism of this combination [144].Ibrutinib appears to downregulate MCL-1 and BCL-xL, potentiating venetoclax's effect [144].Adding venetoclax to obinutuzumab and ibrutinib in combination is being evaluated on a phase Ib study (NCT02427451), and a phase III study (GLOW/CLL3011) is studying ibrutinib plus venetoclax versus obinutuzumab plus chlorambucil (Table 2). BAK, BAX and other pro-apoptotic members are degraded by ubiquitination and the proteasomal pathway.Therefore, proteasomal inhibition allows for their stabilization and accumulation in mitochondria, increasing the pro-to anti-apoptotic protein ratio [145].In relapsed/refractory MM treatment, venetoclax, bortezomib and dexamethasone combination therapy initially showed a high ORR.Patients achieving PR or better had higher levels of BCL-2 [129].However, on the phase III BELLINI study randomizing patients with relapsed/refractory MM to bortezomib combined with venetoclax or matched placebo, although the study showed improved PFS, ORR and uMRD for the venetoclax-containing arm, 13 treatment-emergent deaths occurred in the venetoclax-containing arm.Most deaths were attributable to infection, and this risk strengthened the urge toward a biomarker-driven approach.Authors suggested that this combination could be most relevant in patients with t (11; 14), where a trend towards improved OS was also noted, limiting exposure of toxicity to a smaller group of patients [131,146].An additional phase II study investigating venetoclax with carfilzomib and dexamethasone (NCT02899052) is underway, interim results describe no new safety signals [130]. Strategies inhibiting important cyclin dependent kinases (CDKs) are also promising.CDK9 is a key component of positive transcription elongation factor (pTEFb) which is a transcriptional regulator complex.Inhibition of CDK9 blocks transcription resulting in MCL-1 repression [147].CDK9 inhibition also down-regulates miRNAs that in turn negatively regulates pro-apoptotic BCL-2 family members, leading to a net activation of pro-apoptotic members.Voruciclib, a CDK1, 4, 6 and 9 inhibitor, synergized with venetoclax in DLBCL models to induce tumor remission [148].In MM, several pre-clinical studies have similarly described how CDK inhibition down-regulates MCL-1 in cell lines.In AML, inhibition of CDK9 was demonstrated to transcriptionally silence MCL1, and thus overcome MCL-1 dependent drug resistance [149].In venetoclax-resistant AML cell lines and mouse xenografts, voruciclib combined with venetoclax were synergistic in triggering BIM-dependent apoptosis [150].Several early phase clinical trials investigating combinations of venetoclax with CDK inhibitors are ongoing (Table 2). Promising results are also emerging from the combination of PI3K inhibitors (PI3Ki) with venetoclax and other therapies, particularly in CLL.Recent data released from a phase I/II study of umbralisib (a PI3Ki), ublituximab (a CD-20 antibody) and venetoclax in relapsed/refractory CLL included 27 patients, starting with umbralisib-ublituximab debulking to reduce the risk for tumor lysis syndrome, followed by umbralisib-venetoclax starting from the fourth cycle onwards.In 13 patients treated for >7 cycles of triple combination treatment, the ORR was 100% after cycle 7, and in 9 patients who received 12 or more cycles of treatment, 100% of patients attained uMRD.At short follow up of 6.4 months, none of the 27 patients had experienced disease progression [128] (Table 2). Finally, novel therapies are being combined with BCL-2 inhibition.MDM2 inhibition has been shown to promote MCL-1 degradation in preclinical AML models [151].Early results from a phase Ib study combining idasanutlin with venetoclax in relapsed/refractory AML have shown a response rate of 35.9%, with manageable toxicity [152].Further studies combining venetoclax with novel therapies such as gemtuzumab ozogamicin, enasidenib and liposomal cytarabine and daunorubicin are ongoing (Table 2). ", "section_name": "Promising Combination Strategies in Hematological Malignancies", "section_num": "5.1." }, { "section_content": "Currently, results from targeting the BCL-2 superfamily in solid tumors, using venetoclax or navitoclax have been disappointing [153].On a wide study of multiple solid tumor cell lines, MCL-1 mRNA was the anti-apoptotic BCL-2 member with the highest levels in glioma, lung, renal, prostate, ovarian and breast cancer lines.In comparison, BCL-2 and BFL-1/A1 mRNA levels were highest in leukemia/lymphoma and melanoma cell lines [154].This may explain why therapeutic success with venetoclax monotherapy has been thus far limited to hematological malignancies.Strategies targeting MCL-1 in solid tumors, or combinations including MCL-1 could achieve more success [154].In cervical cancer cell lines, resistance to venetoclax, the BCL-xL selective inhibitor A1331852 or the MCL-1 inhibitor A-1210477 was noted when these agents were used individually.However, combining MCL-1 and BCL-xL inhibitors, or MCL-1 and BCL-2 inhibitors led to inhibition of proliferation in the same cell lines [155]. In other solid tumors, BCL-2 pathway targeting could sensitize to standard therapy, possibly related to its effects on mitochondrial priming.In hormone receptor (HR)-positive breast cancer xenografts, the BH3 mimetics venetoclax and ABT-737 potentiated tumor responses to tamoxifen.Further synergy was seen when the BH3 mimetics were combined with PI3K/mTOR inhibitors, which are already approved therapy in HR-positive advanced breast cancer, in addition to tamoxifen [156].Currently, a randomized phase II study is comparing fulvestrant versus fulvestrant plus venetoclax in advanced HR-positive breast cancer (NCT03584009), and a phase Ib study of combination letrozole, palbociclib and venetoclax in metastatic ER-positive breast cancer is planned (NCT03900884). BH3 mimetics were also shown to potentiate chemotherapy efficacy in basal-like HR-negative breast cancer xenografts.Immunocompromised mouse xenografts were treated with either ABT-737, docetaxel or both [157].As expected, treatment with ABT-737 alone was ineffective, but treatment with combination therapy led to significant improvements in tumor response and OS in-vivo in breast cancer xenografts which overexpressed BCL-2.This finding correlated with a marked increase in apoptosis and BIM-BCL-2 dissociation, and suggests a role for BH3 mimetics to sensitize breast cancers to docetaxel chemotherapy.These results further corroborate with in-vitro experiments showing that endogenous BCL-2 phosphorylation occurs with spindle poison treatment which then leads to increased endogenous BCL-2/BIM binding.The addition of BCL-2 inhibitors was able to disrupt mitotic BCL-2/BIM binding in-vitro, enhancing paclitaxel cytotoxicity [29]. New strategies are exploiting the signaling pathways that induce dependency on BCL-2-like proteins [158].Oncogenic addiction of a cell to RAS, HER2 or EGFR inhibits apoptosis by downregulating BH3-only activator proteins through the MAPK/ERK pathway [158], however, this may also trigger a second oncogenic signal through MYC which promotes BIM expression.Overall, this may lead to increased BCL-2-like protein dependency and increase sensitivity of oncogene addicted cells to apoptosis induced by BH3 mimetics.Two studies have reported on the upregulation of BCL-2-like members in EGFR-TKI-resistant NSCLC which harbor oncogenic EGFR mutations [159,160].In one study, erlotinib-resistant EGFR mutant lung cancer cells showed increased MCL-1 expression, and were sensitive to EGFR TKIs when combined with navitoclax [161]. In melanoma, low BCL-xL expression was shown to bias the anti-apoptotic pool towards MCL-1.The combination of MCL-1 inhibition using AZD5991 with MEK1/2 inhibitors (MEKi) was noted to induce synthetic lethality by BAX/BAK-dependent cell death in-vivo [162].AZD5991 was also shown to delay the development of acquired BRAFi/MEKi resistance, and enhanced the efficacy of ERKi in previously-resistant models [162].Similar observations were made in patient-derived xenograft models of high-grade serous ovarian cancer which were resistant to the MEKi, cobimetinib.Proteomic interrogation showed that cobimetinib upregulated BIM, increasing mitochondrial 'priming', and sensitized models to synergistic targeting with the dual BCL-2/X L inhibitor navitoclax [163].A phase Ib study combining navitoclax with trametinib in RAS-mutant advanced solid tumors is underway (Table 1). ", "section_name": "Targeting BCL2 Pathways in Solid Tumor Therapy", "section_num": "5.2." }, { "section_content": "The recognition of the BCL-2 protein superfamily in regulating intrinsic apoptosis has brought attention to its targeting in overcoming treatment-related resistance in cancer therapy.Progressive refinement in the development of selective BCL-2 inhibitors has led to the successful approval of venetoclax, and significant improvement in clinical outcomes of CLL and AML therapy, while minimizing off-target toxicities.This success has catalyzed the progressive development of other BH3 mimetics, which is likely to change practice in the coming decade.Thus far, the limited success seen in other hematological malignancies and solid tumors only serves to underscore the following challenges we face in harnessing the benefit of BCL-2 inhibitors more broadly. Firstly, though active development of BCL-xL and MCL-1 inhibitors is ongoing, it is uncertain if these inhibitors will maintain sufficient safety profile for widespread use [164].Glaringly, no selective BFL-1/A1 inhibitors have been developed, although the ML214 probe may be useful to evaluate potential interaction sites for BFL-1/A1 inhibition [165].Successful efforts targeting the pro-apoptotic family members are also notably missing from this space; however, apoptotic modulators such as BAM7 which are able to engage the BAX trigger site toward functional oligomerization are under investigation [166]. The selectivity of venetoclax has undeniable benefit in allowing off-target toxicity to be minimized.However, this selectivity itself promotes resistance and compensatory upregulation of non-target anti-apoptotic members such as MCL-1, which may necessitate combination or sequential targeting approaches.At the juncture, it remains to be seen if multiple BH3 mimetics can be successfully used in combination due to overlapping toxicity, and clinical trials evaluating the safety of these combinations are underway. Thirdly, increasing data is emerging regarding the regulatory role that the mitochondrial membrane itself exerts on the BCL-2 superfamily.Membrane insertion and BAX oligomerization are the rate limiting steps for intrinsic apoptosis to proceed.Changes in the mechanical properties of the mitochondrial membrane may regulate BCL-2 proteins, or the membrane itself may have direct effects in modulating BCL-2 family member function [19].One study has reported increased resistance for BAX-BCL-xL complexes when membrane inserted, and it is proposed that the inhibition of BAX oligomerization by BCL-2 proteins in the context of cellular membranes may be an effective means to allow the cell to avoid BAX activation [167].Examining BCL-2 family member interactions in the presence of membranes appears imperative to forward our efforts. The invention of BH3 profiling technology has made it plausible that a means of examining functional BCL-2 protein dependency and its dynamism during cancer development and progression is now available.Its predictive benefit should be consistently evaluated on prospective clinical trials.Additionally, BH3 profiling was developed in and has immense potential in the current era of BH3 mimetics.However, it is not clear if this technology will also help predict benefit to other anti-BCL-2 therapies such as interference strategies [168]. Finally, the BCL-2 family members have numerous non-canonical functions such as cross-talk with metabolic pathways, cellular redox status, involvement in ER calcium homeostasis and autophagy.These pleiotropic effects mean that targeting BCL-2 and BCL-2-like proteins may have a multitude of effects on cancer cell fate, and these consequences on anti-cancer therapy remain under investigation.Recent data have also shown that the BCL-2 family members have an unexpected immunological role, which is provocative for development.In melanoma cells with strong BCL-2 expression, the addition of ABT-737 to co-culture with expanded peripheral blood cytotoxic T-lymphocytes amplified tumor cell kill.As postulated, the addition of BCL-2 inhibitors may sensitize the target tumor cells to perforin/granzyme-B mediated cell kill [169].More recently, Brokatzky and colleagues have described that activation of BAX/BAK induces mitochondrial DNA release [170], which can go on to trigger the innate immune system through the cGAS-STING signaling pathway.This may have the potential to increase the immunogenicity of immunologically \"cold\" tumors.In this way, the mitochondrial apoptosis pathway may serve as a modulator of anti-tumor immunity, therefore paving the way for novel combinations of drugs targeting the BCL-2 family together with immune-checkpoint blockade. ", "section_name": "Future Directions and Challenges", "section_num": "6." }, { "section_content": "Despite the exciting advances made in our understanding of the BCL-2 proteins' role in controlling cell fate and treatment resistance, these observations indicate that the benefit of BCL-2 targeting therapy is not yet fully exploited.The promise of personalized biomarker technology and rational combinations of BCL-2 inhibitors with other branches of cancer therapy are imminent, and will certainly add to our therapeutic arsenal to improve outcomes in a wider group of patients. ", "section_name": "Conclusions", "section_num": "7." } ]	[ { "section_content": "Funding: N.Y.L.N. is supported by the National Medical Research Council, Singapore (MOH-FLWSHP19may-0006), A.L.W. is supported by the National Medical Research Council, Singapore (NMRC/TA17nov003).B.C.G. is supported by the National Medical Research Council, Singapore (NMRC/CSA-SI/0006/2016). S.P. is supported by the National Medical Research Council, Singapore (NMRC CIRG/1433/2015 and OFIRG/0041/2017). ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare no conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.20517/jtgg.2021.08	Genome-wide homozygosity and risk of four non-Hodgkin lymphoma subtypes	Recessive genetic variation is thought to play a role in non-Hodgkin lymphoma (NHL) etiology. Runs of homozygosity (ROH), defined based on long, continuous segments of homozygous SNPs, can be used to estimate both measured and unmeasured recessive genetic variation. We sought to examine genome-wide homozygosity and NHL risk.We used data from eight genome-wide association studies of four common NHL subtypes: 3061 chronic lymphocytic leukemia (CLL), 3814 diffuse large B-cell lymphoma (DLBCL), 2784 follicular lymphoma (FL), and 808 marginal zone lymphoma (MZL) cases, as well as 9374 controls. We examined the effect of homozygous variation on risk by: (1) estimating the fraction of the autosome containing runs of homozygosity (FROH); (2) calculating an inbreeding coefficient derived from the correlation among uniting gametes (F3); and (3) examining specific autosomal regions containing ROH. For each, we calculated beta coefficients and standard errors using logistic regression and combined estimates across studies using random-effects meta-analysis.We discovered positive associations between FROH and CLL (β = 21.1, SE = 4.41, P = 1.6 × 10-6) and FL (β = 11.4, SE = 5.82, P = 0.02) but not DLBCL (P = 1.0) or MZL (P = 0.91). For F3, we observed an association with CLL (β = 27.5, SE = 6.51, P = 2.4 × 10-5). We did not find evidence of associations with specific ROH, suggesting that the associations observed with FROH and F3 for CLL and FL risk were not driven by a single region of homozygosity.Our findings support the role of recessive genetic variation in the etiology of CLL and FL; additional research is needed to identify the specific loci associated with NHL risk.	[ { "section_content": "", "section_name": "", "section_num": "" } ]	[]
10.1186/s13046-019-1076-4	Combination of Enzastaurin and Ibrutinib synergistically induces anti-tumor effects in diffuse large B cell lymphoma	Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma. Although durable remissions can be achieved in more than half of these patients, DLBCL remains a significant clinical challenge, with approximately 30% of patients not being cured. BCR-associated kinases (SYK, BTK, and PI3K) inhibitors have exhibited encouraging pre-clinical and clinical effects, as reported by many researchers. Early studies demonstrated that protein kinase C-β (PKCβ) inhibitors alter phosphorylation level the Bruton's tyrosine kinase (BTK), which leads to enhanced BTK signaling. Here, for the first time, we investigate whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib has synergistic anti-tumor effects in DLBCL.In vitro cell proliferation was analyzed using Cell Titer-Glo Luminescent Cell Viability Assay. Induction of apoptosis and cell cycle arrest were measured by flow cytometry. Western Blotting analysis was used to detect the essential regulatory enzymes in related signaling pathways. RNA-seq was conducted to evaluate the whole transcriptome changes brought by co-treatment with low doses of enzastaurin and ibrutinib. The synergistic anti-tumor effects of enzastaurin and ibrutinib were also evaluated in vivo.Combination of enzastaurin and ibrutinib produced a lasting synergistic effect on the survival and proliferation of DLBCL cells, including reduction of proliferation, promoting apoptosis, inducting G1 phase arrest, preventing cell invasion and migration, and down-regulating activation of downstream signaling. More importantly, whole-transcriptome changes results showed that combination therapy worked synergistically to regulate whole-transcriptome expression compared with enzastaurin and ibrutinib alone. Co-treatment with low doses of enzastaurin and ibrutinib could effectively downregulate BCR, NF-κB, JAK and MAPK related signaling pathway. Furthermore, the mRNA expression analysis further indicated that co-treatment significantly decreased the mRNA levels of NOTCH1. The combination effect in inhibiting proliferation of DLBCL cells probably was realized through suppression of NOTCH1 expression. Finally, the anti-tumor activity of co-treatment also was demonstrated in vivo.Combination of enzastaurin and ibrutinib had synergistic anti-tumor effects in DLBCL, independent of molecular subtype. These results provided a sound foundation for an attractive therapeutic treatment, and the simultaneous suppression of BTK and PKCβ might be a new treatment strategy for DLBCL.	[ { "section_content": "Diffuse large B cell lymphoma (DLBCL), the most common form of lymphoma, is characterized by a heterogeneous tumor entity that can vary in morphologic, biological, immunophenotypic, and clinical presentation, as well as therapeutic outcome [1].Gene expression profiling can be used to differentiate two subtypes of DLBCL, germinal center B-cell like (GCB) and activated B-cell-like (ABC) subgroups of DLBCL, leaving approximately 10~20% of cases \"unclassified\" [1].ABC and GCB DLBCL are characterized by activation of different cellular pathways, posing a major barrier for developing a clear understanding of tumor development, maintenance, and response to therapy [2].Although durable remissions are achieved in more than half of DLBCL patients, the disease remains a major clinical challenge, with approximately 30% of patients not being cured [3].Especially as relapsed/refractory DLBCL patients involve poor survival, novel and effective therapeutic strategies are urgently needed. Abnormal B-cell receptor (BCR) signaling has been implicated in the pathogenesis of B-cell malignancy, which is widely appreciated as one of the primary mechanisms underlying disease progression [4,5].Continuous activation of BCR in ABC-type DLBCL leads to the phosphorylation and activation of regulatory and adaptor proteins, such as spleen tyrosine kinase (SYK), Bruton's tyrosine kinase (BTK), and protein kinase C-β (PKCβ), especially in ABC-type DLBCL [2,[6][7][8].By contrast, oncogenic signaling in GCB DLBCL is typically initiated and reinforced by sharing a dependence on PI3K/mTOR signaling, which is independent of nuclear factor κB (NF-κB) [9,10].In recent years, an increasing number of studies have focused on the therapeutic inhibition of BCR signaling, especially combination-based therapeutic regimens for treating DLBCL [6,11]. Enzastaurin, a potent and selective oral inhibitor of several PKC isoforms, has been shown to regulate the PI3K/ AKT/mTOR, MAPK, and JAK/STAT pathways in solid and hematological malignancies [12][13][14].Although enzastaurin showed promising result in preclinical studies and Phase I/II clinical trials in DLBCL, recent Phase III clinical trials did not meet the primary end point [15][16][17].Interestingly, some researchers have found that PKCβ works as a feedback loop inhibitor of BTK activation, which modulates signaling pathways via altering BTK membrane localization [18,19].PKCβ downregulates BTK activation via both transphosphorylation at Tyr551 and autophosphorylation at Tyr223.Thus, enzastaurin-mediated inhibition of PKCβ leads to enhanced membrane targeting of BTK, increased phosphorylation of PLCγ2, and amplified BCR-mediated Ca 2+ signaling [19]. Ibrutinib is an irreversible small molecule BTK inhibitor that has clearly demonstrated promising therapeutic effects in a variety of B cell malignancies [2,[20][21][22].Therefore, we aimed to investigate whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib has synergistic anti-tumor effects in DLBCL.We demonstrated that low doses of enzastaurin and ibrutinib act synergistically to suppress growth of both ABC and GCB DLBCL cells in vitro and in vivo.These results provide support for future investigation of the combination of enzastaurin and ibrutinib as an attractive therapeutic option for patients with both subtypes of DLBCL. ", "section_name": "Background", "section_num": null }, { "section_content": "Cell lines and cell culture HBL-1, TMD8, OCI-LY7 cell lines were generously provided by Dr. Fu, University of Nebraska Medical Center (Omaha, NE, USA).SU-DHL-2 and SU-DHL-6 cells were obtained from American Type Culture Collection (Manassas, VA, USA).Cells were grown in RPMI 1640 medium (Gibco, Life Technologies, CA, USA) supplemented with 10-20% fetal bovine serum (Gibco, Life Technology, CA, USA), penicillin/ streptomycin, glutamine, beta-mercaptoethanol. Except for OCI-LY7, which was maintained in IMDM (Gibco, Life Technology, CA, USA) supplemented with beta-mercaptoethanol, penicillin/ streptomycin, and 20% heparinized human plasma.All cell lines were maintained in a humidified 5% CO 2 incubator at 37 °C.Identification of all DLBCL cell lines was confirmed by short tandem repeat DNA fingerprinting analysis (Applied Biosystems, Foster City, CA, USA). ", "section_name": "Methods", "section_num": null }, { "section_content": "Enzastaurin was a gift from Denovo Biopharma (San Diego, USA), and ibrutinib was purchased from Medchem Express (NJ, USA).It was initially dissolved in 100% DMSO (Sigma-Aldrich, Darmstadt, Germany) at a concentration of 10 μM and stored in -80 °C.Primary and secondary antibodies were listed in additional file (Additional file 1: Table S1). ", "section_name": "Drugs and reagents", "section_num": null }, { "section_content": "Cells were seeded in a 96-well culture plate at a density of 3000 cells per 100 μl and treated with different concentrations of enzastaurin and ibrutinib for 72 h.Cells were counted and viability was assessed using Cell Titer-Glo Luminescent Cell viability assay system (Promega, Madison, WI, USA).Luminescent signals were measured by LMax II (Molecular Devices, Sunnyvale, CA, USA).Inhibition rates were calculated following the formula: inhibition rates = (1-dosing/control) × 100%. ", "section_name": "Analysis of cell proliferation", "section_num": null }, { "section_content": "Cells were treated with vehicle or indicated concentrations of enzastaurin and ibrutinib for 48 h for apoptosis and cell cycle analysis.For apoptosis assays, cells were stained with annexin V-APC (Biolegend, CA, USA) according to the protocol.For cell cycle assays, cells were stained with PI staining buffer (Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer's protocol.Finally, the labeled cells were analyzed using BD Accuri C6 flow cytometer (BD, Biosciences, San Jose, CA). ", "section_name": "Apoptotic cells and cell-cycle assays", "section_num": null }, { "section_content": "Total cellular RNA was extracted using Trizol reagent (Life Technologies, Carlsbad, CA) and cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China).qRT-PCR analysis was performed using Go Taq qPCR Master Mix (Promega Corporation, Madison, USA).Specific primers for NOTCH1 (Forward: 5′-TCCACCAGTTTGAATGGTCAAT-3′; Reverse: 5′-CGCAGAGGGTTGTATTGGTTC-3′) and GAPDH (Forward: 5′-GCACCGTCAAGGCTGAGAAC-3′; Reverse: 5′-TGGTGAAGACGCCAGTGGA-3′) were used to perform qRT-PCR.All reactions were run in Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Woburn, MA, USA), mRNA expression data were calculated using the following equation: RQ = 2 -ΔΔCt . ", "section_name": "Real-time reverse transcription-PCR (qRT-PCR) assay", "section_num": null }, { "section_content": "Harvested cultured cells were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA) with protease/ phosphatase inhibitor (Roche, Mannheim, Germany).Signaling proteins were detected by western blot as previously described [23].Immunopositive bands were visualized using chemiluminescence detection system (Alpha Innotech, San Leandro, CA, USA) according to the manufacturer's instructions. ", "section_name": "Western blotting and signaling assays", "section_num": null }, { "section_content": "Cells were treated with vehicle or indicated concentrations of enzastaurin and ibrutinib for indicated time in FBS-free RPMI 1640.For cell invasion assays, cells were placed into Matrigel basement membrane matrix-coated upper chambers in a transwell plate with 8.0 μM pores (Corning Costar, NY, USA).For cell migration assays, cells were seed into transwell with 8.0 μm pore polycarbonate membrane insert (Corning Costar, NY, USA).The lower portion of the chamber contained 30% FBS for use as a chemoattractant.After 24 h (48 h), the number of cells migrating (invading) into the lower chamber were counted using Cell Titer-Glo Assays.Invasive and migration abilities were determined by the number of viable cells in the lower chamber. ", "section_name": "Invasion and migration assay", "section_num": null }, { "section_content": "Cells were treated with the indicated drug alone or in combination for 24 h, and then total RNA was isolated.Total RNA (3 μg) was converted to cDNA using TransScript First-Strand cDNA Synthesis SuperMix.RNA quantification and qualification, library preparation, clustering and sequencing, read mapping and data processing were performed in Novogene Bioscience (Beijing, China).Differential expression analysis of two groups (two biological replicates per group) was performed using the DESeq2 R package (1.16.1).Corrected P-value of 0.05 and absolute foldchange of 2 were set as the threshold for significantly differential expression.To analyze the underlying mechanism of the sets of genes which were differentially expressed following each treatment, we used clusterProfiler R package to test the statistical enrichment of differential expression genes in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. ", "section_name": "Gene expression profiling and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis", "section_num": null }, { "section_content": "Lentiviral vectors (GV493) containing green fluorescent protein (GFP) (shControl) or NOTCH1-specific short hairpin RNA (shNOTCH1, sequence #1: 5′-TGCCAACATCCAGG ACAACAT-3′) were constructed, packed, and purified by Genechem (Shanghai, China).Cells were infected with shControl, shNOTCH1, at MOI 1: 100 and cultured for > 72 h to be used for the downstream experiments.The depletion efficiency was assessed by western blot analysis. ", "section_name": "Lentivirus packing and infection", "section_num": null }, { "section_content": "All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals and in accordance with the ethical guidelines of CrownBio (Beijing, China).Female immune-deficient NPG mice (NOD-Prkdcscid Il2rgnull), six to eight weeks old, were obtained from HFK Bioscience Co.Ltd.(Beijing, China).HBL-1 tumor cells (5 × 10 6 ) in serum-free medium with matrigel (1:1 ratio) were injected subcutaneously into the area under the right flank of each mouse.When the tumor reached 100-150 mm 3 , mice were randomly divided into four groups (control, treated with enzastaurin, treated with ibrutinib, treated with both enzastaurin and ibrutinib).Enzastaurin (125 mg/kg, dissolved in 10% Acacia) was administered twice daily orally and/or ibrutinib (12 mg/kg, dissolved in 1% methylcellulose, 0.4% Cremophor® EL) was administered once daily orally for 21 days.Tumor volume (V) and body weight were monitored two to three times per week.The tumor volume (V) was calculated as V = (length×width 2 ) /2. Tumor tissue samples were collected from all groups at 4 h after the last dose. ", "section_name": "Detection of treatment efficacy in vivo", "section_num": null }, { "section_content": "TUNEL is a method for detecting DNA fragmentation by labeling the 3′-hydroxyl termini in the double-strand DNA breaks generated during apoptosis.HBL-1 tumor samples were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5 μm sections.A TUNEL assay was then conducted to examine DNA fragmentation using an in situ cell death detection kit (Cat No. 11684795910, Roche, Mannheim, Germany) according to the manufacturer' s instructions. ", "section_name": "In situ apoptosis quantification by TUNEL", "section_num": null }, { "section_content": "Immunohistochemistry stains for Ki-67, p-BTK and p-PKCβ were performed in the department of pathology of Peking University Cancer Hospital using the standard streptavidin-biotin-peroxidase immunostaining procedure.The slides were incubated with primary antibody overnight at 4 °C and then with HRP-conjugated secondary antibody at room temperature for 30 min.DAB was used for staining.The intensity and density of the staining were examined in a double-blinded manner by two independent pathologists from the department of pathology in Peking University Cancer Hospital & Institute.Primary antibodies were listed in Additional file 1: Table S1. ", "section_name": "Immunohistochemistry", "section_num": null }, { "section_content": "All experiments in vitro were independently done more than three times.The SPSS 22.0 statistical software (IBM, New York, NY, USA) was used for all analyses.Data were analyzed using paired or unpaired Student's t test comparisons or one-way ANOVA.P values <0.05 were accepted as statistically significant.The combination index (CI) for drug combination was determined according to the Chou-Talalay method using the CalcuSyn software (version 2, Biosoft, Cambridge, UK).CI values <1, =1, and > 1 indicates synergism effects, additive effects, and antagonism effects, respectively. ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "To determine the effect of enzastaurin on the survival of DLBCL cell lines, we cultured nine cell lines in the presence of enzastaurin (0 to 20.0 μM) for 72 h.As shown in Fig. 1a, treatment with enzastaurin resulted in a dose-dependent inhibition of cell proliferation, with a 50% inhibitory concentration (IC50) values ranging between 6.7 and 15.6 μM (Fig. 1a).We confirmed that treatment with enzastaurin effectively reduced the viability of DLBCL cells, and there was no statistical difference between ABC and GCB cells lines (p = 0.48). PKCβ is a common signaling target that lies downstream of BTK.Surprisingly, we observed that HBL-1 and TMD8 cells exhibited notable upregulation of phosphorylated BTK (p-BTK) upon treatment with enzastaurin (Fig. 1b).These results suggest that although inhibition of PKCβ is therapeutically effective in DLBCL cells, it also leads to positive regulation of BCR signal pathway.Thus, while pharmacological inhibition of enzastaurin attenuated some branches of BCR signaling pathways, inactivation of these pathways can be compensated by upregulation of other pathways (Fig. 1c).These compensatory pathways greatly limit the effectiveness of enzastaurin in DLBCL, especially as a monotherapy. ", "section_name": "Enzastaurin inhibited proliferation of ABC and GCB cell lines in a dose-dependent manner and upregulates BTK phosphorylation", "section_num": null }, { "section_content": "Our initial results suggested that simultaneous inhibition of PKCβ and BTK would block BCR signaling and induce cell death in DLBCL cells.Based on the cytotoxicity of enzastaurin and ibrutinib, we exposed the GCB (SU-DHL-6 and OCI-LY7) and ABC (HBL-1, TMD8 and SU-DHL-2) lymphoma cells to minimally toxic concentration of enzastaurin, together with increasing concentrations of ibrutinib in combination for 72 h.The toxicity of each treatment was assessed by measuring the rate of growth inhibition.Notably, DLBCL cells (SU-DHL-2 and SU-DHL-6) that responded poorly to enzastaurin or ibrutinib as a single-agent therapy were exquisitely sensitive to combination treatment with these two drugs (Fig. 2a).Combination therapy with enzastaurin and ibrutinib greatly increased the inhibition rate of DLBCL cell growth irrespective of the molecular subtype or the level of responsiveness to ibrutinib monotherapy (Fig. 2a). To further confirm the synergistic effect of enzastaurin and ibrutinib in DLBCL, CI values were calculated (Fig. 2b).The combined therapy showed a strong synergistic inhibitory effect on the growth of HBL-1, TMD8, SU-DHL-2, SU-DHL-6 and OCI-LY7 cells at all tested doses, with CI value ranging from 0.239 to 0.686.The synergistic effects in SU-DHL-2 were weak, with a CI range of 0.608-0.923.Overall, the combinations of enzastaurin and ibrutinib thus exhibited synergistic effects in GCB and ABC subtypes of DLBCL cell lines at all doses examined (CI < 1, Fig. 2b). Time-course analysis of cell death further indicated that that prolonged exposure to the combination had an even greater effect on inhibition of cell proliferation (Fig. 2c).Thus, the combination of enzastaurin and ibrutinib demonstrated long-term synergistic effects on the survival and proliferation of DLBCL cells, independent of their subtype. ", "section_name": "Synergistic effects of enzastaurin and ibrutinib on the induction of cell death in DLBCL cell lines", "section_num": null }, { "section_content": "To determine whether inhibition of cell growth by co-treatment with enzastaurin and ibrutinib was associated with apoptosis and/or cell cycle arrest, we analyzed levels of apoptosis in four cells lines after 48 h exposure to the indicated concentrations of enzastaurin and/or ibrutinib.In HBL-1, the combination of enzastaurin with two different doses of ibrutinib induced 43.8 ± 8.7% or 51.4 ± 5.9% apoptosis respectively, as measured by annexin V staining; these values were greater than those cells treated with each single agent alone (enzastaurin = 25.5 ± 5.4%, ibrutinib = 15.9 ± 6.0% and 19.0 ± 6.7%, Fig. 3a).Thus, co-treatment with enzastaurin and ibrutinib has a synergistic effect on promoting apoptosis.Consistent with the results of annexin V staining, expression of proteins associated with apoptosis also changed in response to co-treatment in HBL-1 cells (Fig. 3b).Treatment with either enzastaurin or ibrutinib slightly increased expression the active forms of poly-ADP ribose polymerase (PARP) and caspase-3, but co-treatment dramatically increased these effects (Fig. 3b).Treatment with the combination also induced a sharp decrease in the expression of anti-apoptotic Bcl-2 family members, including Mcl-1, XIAP, and Bcl-2.Similar results were observed in TMD8, SU-DHL-6 and OCI-LY7 cells (Fig. 3a,b).Taken together, these results show that the con-administration of enzastaurin and ibrutinib promotes apoptosis through activation of the caspase-dependent and mitochondrial pathway in DLBCL cells, ultimately resulting in cytotoxicity. In order to assess the effects of enzastaurin and ibrutinib on the cell cycle, we used flow cytometry to analyze the cell cycle profiles of treated cells (Fig. 3c).The percentage of HBL-1 cells in G1 phase increased from 28.5 ± 0.05% in the control group to 46.4 ± 0.84% and 47.2 ± 3.12% in the combination treatment groups.A corresponding decrease of cells in S phase also occurred.Similar results were observed in TMD8, SU-DHL-6 and OCI-LY7 cells (Fig. 3c).Consistent with these results, expression of CDK2, CDK4, CDK6 and Cyclin D1 substantially decreased in cells co-treated with enzastaurin and ibrutinib, whereas treatment with single agents only mildly affected the expression of these proteins known to play essential roles in the G1/S transition.Similar trend were observed in the other three cell lines (Fig. 3d).These data demonstrate that the combinations of enzastaurin and ibrutinib induced G1 phase arrest and the combination therapy suppressed cell proliferation by inducing both cell cycle arrest and initiation of apoptotic. ", "section_name": "The combination of enzastaurin and ibrutinib promoted apoptosis and induced G1 arrest in DLBCL cells", "section_num": null }, { "section_content": "In order to assess the possible effects of treatment with low doses of enzastaurin and ibrutinib on cell motility, we performed cell migration and invasion assays using DLBCL cells.For invasive abilities, treatment with enzastaurin or ibrutinib alone slightly suppressed invasive of HBL-1 cells, with 97.0 and 85.0% cells exhibiting invasion, respectively.In contrast, invasion was notably suppressed in cells treated with the combination of enzastaurin and ibrutinib, with only 32.8% of cells invading relative to the control group (Fig. 4a).Analysis of migration revealed that treatment with the single agent reduced migration to 79.0 and 70.2% of HBL-1 cells, respectively.In contrast, the number of co-treated cells passing through the membrane was only approximately 25.5% of the control cells (Fig. 4b).Similar trends were observed in TMD8, SU-DHL-6 and OCI-LY7 cells, and detailed results are shown in the invasion and migration histogram (Fig. 4c).These findings demonstrate that enzastaurin and ibrutinib synergistically decrease cell migration and invasion, which are essential for DLBCL cell motility. ", "section_name": "Treatment with low doses of enzastaurin and ibrutinib synergistically inhibits migration and invasion in DLBCL", "section_num": null }, { "section_content": "To gain insight into the mechanism underlying the anti-proliferative effects of co-treatment with enzastaurin and ibrutinib in DLBCL models, we next investigated the changes of signal transduction pathways in treated cells.As shown in Fig. 5a, HBL-1 cells treated with low doses of enzastaurin monotherapy for 60 min and 120 min showed clearly reduced the phosphorylation of glycogen synthase kinase 3β (GSK3β), which serves as a biomarker for enzastaurin activity.Short-term and low-dose enzastaurin treatments had not significantly affect on the PKCβ phosphorylation (data not show), and increased expression of p-BTK, p-ERK.Similarly, treatment with Ibrutinib alone reduced levels of BTK phosphorylation, which was accompanied by a mild effect on phosphorylation of mTOR, PLCγ2, and ERK.However, co-treatment with enzastaurin and ibrutinib resulted in a greater reduction in phosphorylation of ERK, mTOR, PLCγ2, compared to each monotherapy alone (Fig. 5a,b).These results were also confirmed in TMD8 and SU-DHL-6 cells (Fig. 5b, Additional file 2: Figure S1).Overall, in contrast to single treatment, the combination of enzastaurin and ibrutinib appears to more effective inhibit signal transduction in both ABC and GCB cell models, indicating that co-treatment successfully suppress multiple signaling pathways downstream of BCR. ", "section_name": "Co-demonstration of enzastaurin and ibrutinib synergistically inhibit downstream signaling pathways", "section_num": null }, { "section_content": "In order to better understand the effects of combination therapy with low doses of enzastaurin and ibrutinib in DLBCL cells, we assayed whole-transcriptome changes by RNA-sequencing.Several hundred transcripts observed to be either up or down regulated by different treatments.Because the upregulated genes were not closely associated with these inhibitors, only the downregulated genes were further analyzed.Venn diagram was used to depict the number of downregulated genes associated with the different treatments (< 2 fold, p < 0.05).Enzastaurin and ibrutinib were less efficient as single agents, with 399 and 336 transcripts significantly downregulated, respectively, compared with 605 downregulated transcripts for the combination treatment.Approximately 91% of transcripts (365 genes) repressed by enzastaurin and 73% of transcripts (246 genes) repressed by ibrutinib were included in the combination group.Additionally, the combination treatment efficiently downregulated an additional 163 transcripts that had not been downregulated by either drug alone.Similar results were observed in TMD8 cells (Fig. 6a).Thus, co-treatment with enzastaurin and ibrutinib resulted in the downregulation of a broader set of genes compared to the treatment with either of the compounds alone. Further analysis of downregulated genes showed that compared with vehicle treatment control, significantly downregulated genes from top ranked pathways (by KEGG) are represented in the heat map (Fig. 6b).Co-treatment with low doses of enzastaurin and ibrutinib effectively downregulated genes associated with BCR, NF-κB, JAK-STAT and MAPK signaling pathways.These pathway analysis results were also confirmed in TMD8 cells (Additional file 2: Figure S2), which consistent with those from Western blot results (Figs. 3,5).Thus, combination therapy appeared to synergistically regulate whole-transcriptome changes. To further assess the synergistic anti-tumor effects of enzastaurin and ibrutinib, we analyzed the expression of transcripts downregulated by the combination treatment using qRT-PCR.Compared with enzastaurin and ibrutinib monotherapy, combination treatment was able to decrease the mRNA expression of NOTCH1 more significantly (Fig. 6e).A strong body of evidence underscores the important oncogenic role of NOTCH1 in promoting changes in cellular metabolism, cell growth and proliferation, and enhanced the activity of signaling pathways [23][24][25][26].Furthermore, aberrant NOTCH1 activity has emerged as an important oncogenic regulator of hematological malignancy [26].The NOTCH1 mRNA and protein were expressed at medium-to-high levels in DLBCL cells (Fig. 6c).Thus, the anti-proliferative effects of the combination of enzastaurin and ibrutinib in DLBCL cells are likely due to suppression of NOTCH1 expression. To validate the role of NOTCH1 downregulation in DLBCL cell survival and proliferation, we used shRNA transfection to knock-down NOTCH1expression (Fig. 6d).Silencing of NOTCH1 in DLBCL cells had anti-proliferative effects, indicating that NOTCH1 expression is important for the survival of DLBCL cells.Similar proliferation effects and timing were observed in NOTCH1 shRNA treatment and co-treatment with enzastaurin and ibrutinib, suggesting that the synergistic effects of the combination treatment may occur through downregulating NOTCH1 expression (Fig. 6f). ", "section_name": "Whole-transcriptome changes in DLBCL occur in response to the combination of enzastaurin and ibrutinib", "section_num": null }, { "section_content": "Finally, we assessed the ability of enzastaurin, alone and in combination with ibrutinib, to reduce tumor growth in a lymphoma model, in which ABC-DLBCL HBL-1 cells were engrafted in NPG mice (Fig. 7).Enzastaurin or ibrutinib monotherapy resulted in a smaller reduction in tumor volume relative to the control.Compared with control and monotherapy, tumor volumes were significantly smaller in mice treated with the combination treatment (p < 0.05, Fig. 7a).Treatment was well tolerated, and no mice lost weight obviously or died (Fig. 7b).At the end of the experiment, neither enzastaurin (811.28 ± 182.10 mg) nor ibrutinib (719.25 ± 156.71 mg) significantly inhibited tumor growth compared with that of the vehicle group (1075.29 ± 152.56 mg), while the co-treatment robustly suppressed tumor growth and restrained tumor weight (444.65 ± 87.64 mg, Additional file 2: Figure S3).To further evaluate the apoptosis, proliferation, and BCR signal status of tumor tissue post different treatments, TUNEL, Ki-67, p-BTK and p-PKCβ was investigated and quantified in paraffin sections of tumor samples collected from HBL-1 xenografts.As shown in Fig. 7c andd, combination therapy of enzastaurin and ibrutinib induced a notable increase of apoptosis compared with each agent alone.Moreover, co-treatment with enzastaurin and ibrutinib produced a more significant decrease of Ki-67, p-PKCβ and p-BTK expression than the monotherapy achieved (Fig. 7e,f).Thus, these results demonstrate that the co-treatment of enzastaurin with ibrutinib has synergistic activity in preclinical models, confirming our in vitro findings. ", "section_name": "Enzastaurin and ibrutinib have synergistic antitumor effects in a DLBCL models in vivo", "section_num": null }, { "section_content": "DLBCL is a heterogeneous lymphoma, and although the introduction of rituximab has greatly improved clinical outcomes, it still proves incurable in 30%~40% of all cases [27].One of the most important reasons underlying negative outcomes is that ABC and GCB DLBCLs exhibit activation of different signaling pathways.The ABC subtype is characterized by mutations in MYD88, CARD11, CD79A and CD79B, and constitutive activation of NF-κB signaling, features associated with less favorable clinical outcome [6,7].In contrast, GCB subtype is more frequently characterized by activation of the PI3K/AKT pathway, rather than NF-κB pathway [10].These differences in signaling translate into different levels of tumor aggressiveness and differential response to therapy [28].The crucial role played by the BCR signaling pathways in DLBCL has prompted the development of targeted kinase inhibitors, including inhibitors of BTK, PI3K, SYK and PKCβ, representing promising potential therapeutic strategies for DLBCL patients [29].Here, for the first time, we demonstrate that combination treatment with enzastaurin and ibrutinib augments anti-tumor effects of the single agents in DLBCL in vitro and in vivo.These effects may be due to inactivation of related signaling pathways and downregulation NOTCH1 expression. Enzastaurin is a relatively well-studied anti-tumor agent.Preclinical evaluation of enzastaurin has shown promising results in cutaneous T-cell lymphoma, B-cell lymphoma, multiple myeloma (MM), Waldenstrom's macroglobulinemia (WM) and other solid tumors [30][31][32].With respect to DLBCL, 22% of DLBCL tumor samples have been found to be positive for PKCβ expression, defined as immunostaining of > 50% of cells [33].Furthermore, PKCβ expression is a useful marker of poor prognosis in DLBCL.Phase I/II studies of enzastaurin have shown that it is well tolerated in DLBCL patients, 15% (8/55) of the patients experienced prolonged freedom from progression (FFP ≥ 4 cycles) and 7% (4/55) of patients experienced FFP (See figure on previous page.)Fig. 3 The combination of enzastaurin and ibrutinib promoted apoptosis and induced G1 phase arrest.a Combination treatment prompted apoptosis in DLBCL cells.Cells pre-treated with indicated concentrations of ibrutinib in the presence or absence of fixed concentration of enzastaurin for 48 h were stained with annexin V-APC, then apoptosis was assessed using flow cytometry.Apoptosis cells were determined by APC + cells.b The combination treatment mediated expression of proteins associated with apoptosis in DLBCL cells.After 48 h of exposure to enzastaurin and/or ibrutinib in combination, proteins were extracted from cells of different groups and proteins associated with apoptosis were analyzed by western blot.c Co-treatment with enzastaurin and ibrutinib induced G1 phase arrest in DLBCL cells.Cells were treated with different concentrations of ibrutinib in the presence or absence of fixed concentration of for enzastaurin 48 h, and then stained with propidium iodide (PI).Cell cycle was assessed using flow cytometry.d The combination treatment group mediated alterations in proteins associated with G1/S transition in DLBCL cells.After 48 h of exposure to indicated concentration of enzastaurin and ibrutinib in combination, proteins were extracted from cells of different groups and proteins were analyzed by western blot.Error bars represent the result and SD of three different experiments.* p < 0.05, p < 0.01, * p < 0.001, compared with control group; # p < 0.05, ## p < 0.01 compared with enzastaurin group 20~50 months [16,17].However, similar results have not be observed in a phase III clinical trial (PRELUDE), which showed that enzastaurin monotherapy did not significantly improve disease-free survival (DFS) in high-risk DLBCL patients after remission of B cell lymphoma.These results have essentially halted the development of enzastaurin as a monotherapy in DLBCL.A large phase 3 global clinical trial was launched to assess enzastaurin plus R-CHOP in DLBCL patients with the genomic biomarker DGM1, identifying a novel genetic biomarker related to drug efficacy, which could improve the efficacy and outcomes of enzastaurin. Analysis of failed therapeutics presents an opportunity for improvement through both preclinical and clinical investigations of therapeutic combinations.Prior studied have noted the combination treatment with HDACi and enzastaurin exhibit a synergistic effect in DLBCL.HDACi may increase the expression of PKCβ, leading to activation of survival signals [14].Additionally, therapeutic regimens composed of enzastaurin with other agents, such as lenalidomide, NVP-BEZ235 (PI3K inhibitor), and bortezomib, have been evaluated in non-Hodgkin lymphoma B-cell lines [13,34,35].These studies can be considered as examples of new innovative attempts to identifying logical therapeutic combination. Ibrutinib (PCI-32765) is an orally active inhibitor of BTK that binds Cysteine-481 on the kinase domain, leading to an irreversible inhibition at Tyr-223.Remarkable progress has been made in the development of ibrutinib in recent years, and the drug demonstrated considerable efficacy in a variety of B-cell malignancies.In ABC and GCB DLBCL, differences in activation of signaling pathways translate to differences in response to BTK inhibition, which have largely been confirmed in a Phase II trial of ibrutinib in relapsed DLBCL patients.Results from this trial revealed an overall response rate (ORR) of 37% (14/38) of patients with ABC-DLBCL, but only 5% (1/20) of patients with GCB-DLBCL [2].Furthermore, ABC-DLBCL patients harboring CD79A/B mut , CARD11 mut , TNFAIP3 mut , or MYD88 mut showed primary resistance to ibrutinib [2,6,36].On the other hand, as a result of activating mutations in BTK or PLCγ2, a subset of patients with an initially response to ibrutinib eventually relapse, underscoring the need for developing new target agents and combination treatments to improve the outcomes of such resistant patients [37].Recent attention has been focused on potential drug combinations in DLBCL, particularly co-treatment with a BTK inhibitor and lenalidomide, bortezomib, PI3K inhibitor, or Pan-SRC kinase inhibitors in DLBCL [6,29,[38][39][40].Addition of ibrutinib to DLBCL cells treated with these agents resulted in synergistic cytotoxic effects on cells.There is also clinical data supporting the use of ibrutinib in combination with other agents, as combination therapy with rituximab and ofatumumab has been shown to be effective for the treatment of relapsed or refractory CLL/SLL [41].Current on-going trials will further define the role of ibrutinib as upfront therapy and/ or as a combination treatment in B-cell lymphoid malignancies.In the present study, analysis of the combination of PKCβ inhibitor enzastaurin and the BTK inhibitor ibrutinib has shown synergistic anti-tumor effects in DLBCL, thereby providing a rational basis for future preclinical/ clinical investigations that may allow for the development of specific, well tolerated and efficient cancer therapeutics for relapsed or refractory DLBCL patients. Another critical reason for supporting the combination treatment of enzastaurin with ibrutinib is that early studies have demonstrated the role of PKCβ in the negative regulation of BTK.Also, treatment with PKCβ inhibitors alter phosphorylation of BTK, leading to enhanced BTK signaling [18,19].Consistent with these previous works, our study also revealed that the expression of p-BTK was markedly increased by treatment of enzastaurin.Thus, PKCβ potently activates negative feedback signals of BTK, indicating that PKCβ inhibitors upregulate BTK's activation thereby altering oncogenic signals downstream of BCR.Based on this mechanism, we investigated whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib had synergistic anti-tumor effects in DLBCL.Our findings revealed synergistic effects of these two agents on reduction of proliferation, promoting apoptosis, inducting G1 phase arrest, inhibition of cell invasion and migration, and downregulation activation of downstream signaling in GCB and ABC lymphoma cell lines. Combination treatment of enzastaurin with ibrutinib has also been shown to trigger time-dependent inhibition of NOTCH1 mRNA expression, whereas treatment with either drug alone only slightly affected NOTCH1expression.The oncogenic role of NOTCH1 has been verified in a number of hematological diseases, including T-cell acute lymphoblastic leukemia, multiple myeloma, Hodgkin and anaplastic large cell lymphoma [23,24,26].Many recent studies also have shown that a large number of DLBCL patients harbor NOTCH1 mutations and aberrations, validating the oncogenic role of NOTCH1 as the genetic drivers of DLBCL [42][43][44].Moreover, NOTCH1 promotes the activation of the PI3K-AKT-mTOR and NF-κB signaling pathway, which plays a pivotal role in accelerating cell growth and promoting cell apoptosis not only in T-cell (See figure on previous page.)Fig. 6 Whole-transcriptome changes in DLBCL occur in response to the combination of enzastaurin and ibrutinib.HBL-1 and TMD8 cells were exposed for 24 h with enzastaurin (HBL-1 2 μM, TMD8 4 μM) and/or ibrutinib (HBL-1 0.02 μM, TMD8 0.003 μM).RNA was collected for RNA sequencing.a Venn diagram illustrating the number of overlapping downregulated gene between different groups.b Significantly downregulated genes from top ranked pathways (by KEGG) are represented in the heatmap.Colors scale bar represents from higher (red) to lower (blue) expression.Gene expression levels are expressed in FPKM values, differences shown in color scale after Z-score transformation.Downregulated genes were determined by log2foldchange<0.FPKM, fragments per kilo base of exon per million fragments mapped.c The expressions of NOTCH1 in DLBCL cell lines were detected using qRT-PCR and western blot.d NOTCH1 knockdown by shRNA was validated by western blot in HBL-1, TMD8, OCI-LY7, SU-DHL-6 cells.β-actin is shown as a loading control.e The expressions of NOTCH1 gene were further confirmed in DLBCL cells after pre-treated with enzastaurin and/or ibrutinib.f The DLBCL cells were transfected with shRNA targeting NOTCH1, or treated with enzastaurin and ibrutinib for 48 h, 72 h.The cell viability of tumor cells was determined using the Cell Titer-Glo luminescent cell viability assay.Results are expressed as mean ± SD, data are representative of three independent experiments.* p < 0.05, p < 0.01, * p < 0.001 compared with control group; # p < 0.05, ## p < 0.01 compared with enzastaurin group but also in B-cell neoplasms [24,25].In our works, treatment of DLBCL with a combination of enzastaurin and ibrutinib significantly reduced expression of NOTCH1, and shRNA mediated reduction in NOTCH1 expression dramatically inhibited DLBCL cell proliferation.These data indicated that downregulation of NOTCH1 could be a crucial biological mechanism by which the synergistic effect of co-treatment with enzastaurin and ibrutinib in suppressing cell growth.The precise mechanism in detail is likely to be a promising direction of further research. ", "section_name": "Discussion", "section_num": null }, { "section_content": "We have evaluated the combination of enzastaurin and ibrutinib in DLBCL in vitro and in vivo, demonstrating the co-treatment had synergistic anti-tumor effects in DLBCL, independent of molecular subtype.These results provide a sound foundation for further evaluation of an attractive therapeutic combination, suggesting that simultaneous inhibition of BTK and PKCβ may represent a novel, effective therapeutic approach for ABC and GCB DLBCL. ", "section_name": "Conclusions", "section_num": null } ]	[ { "section_content": "We thank Dr. Fu from the University of Nebraska Medical Center in USA for the kind gifts of the DLCBL cell lines.We also thank Dr. Luo Wen from Denovo Biopharma for provide guidance for experimental design, Dr. Huirong Ding and Dr. Xijuan Liu from the Peking University Cancer Hospital & Institute for the analysis of flow cytometry.Further thanks are due to Drs Yunfei Shi and Bin Dong from Peking University Cancer Hospital & Institute for analysis of Immunohistochemistry stains. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "This work was financially supported by NSFC (Nos.81870154, 81470368, 81670187 and 81600164), Beijing Natural Science Foundation (No. 7172047 and 7172046), Capital's Funds for Health Improvement and Research (No. 2018-1-2151), Beijing Municipal Administration of Hospitals' Ascent Plan (No. DFL20151001), Beijing Municipal Administration of Hospitals Clinical Medicine Development of special funding support (No. XMLX201503) and Beijing Municipal Administration of Hospitals Incubating Program (Code.PX2017001). ", "section_name": "Funding", "section_num": null }, { "section_content": "The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. ", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "Additional file 1: Table S1. This study was approved by review board of the Peking University Cancer Hospital & Institute.Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, which were approved by the Peking University Cancer Hospital & Institute. Not applicable. The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Additional files", "section_num": null }, { "section_content": "Additional file 1: Table S1. ", "section_name": "Additional files", "section_num": null }, { "section_content": "This study was approved by review board of the Peking University Cancer Hospital & Institute.Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, which were approved by the Peking University Cancer Hospital & Institute. ", "section_name": "Ethics approval and consent to participate", "section_num": null }, { "section_content": "Not applicable. ", "section_name": "Consent for publication", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.3390/genes15030355	A POT1 Founder Variant Associated with Early Onset Recurrent Melanoma and Various Solid Malignancies	<jats:p>POT1 (Protection of Telomeres 1) is a key component of the six-membered shelterin complex that plays a critical role in telomere protection and length regulation. Germline variants in the POT1 gene have been implicated in predisposition to cancer, primarily to melanoma and chronic lymphocytic leukemia (CLL). We report the identification of POT1 p.(I78T), previously ranked with conflicting interpretations of pathogenicity, as a founder pathogenic variant among Ashkenazi Jews (AJs) and describe its unique clinical landscape. A directed database search was conducted for individuals referred for genetic counselling from 2018 to 2023. Demographic, clinical, genetic, and pathological data were collected and analyzed. Eleven carriers, 25 to 67 years old, from ten apparently unrelated families were identified. Carriers had a total of 30 primary malignancies (range 1–6); nine carriers (82%) had recurrent melanoma between the ages of 25 and 63 years, three carriers (27%) had desmoid tumors, three (27%) had papillary thyroid cancer (PTC), and five women (63% of female carriers) had breast cancer between the ages of 44 and 67 years. Additional tumors included CLL; sarcomas; endocrine tumors; prostate, urinary, and colorectal cancers; and colonic polyps. A review of a local exome database yielded an allelic frequency of the variant of 0.06% among all ethnicities and of 0.25% in AJs. A shared haplotype was found in all carriers tested. POT1 p.(I78T) is a founder disease-causing variant associated with early-onset melanoma and additional various solid malignancies with a high tumor burden. We advocate testing for this variant in high-risk patients of AJ descent. The inclusion of POT1 in germline panels for various types of cancer is warranted.</jats:p>	[ { "section_content": "Telomeres are regions composed of repetitive nucleotide sequences that are located at the ends of linear chromosomes.They are directly involved in protecting the chromosomal ends from enzymatic degradation and from accidental DNA recombination during cellular division.With each cellular division, telomeres shorten.This phenomenon plays a pivotal role in the aging process and cellular lifespan.The telomerase enzyme synthesizes new telomere repeats with each cycle to maintain telomere length and promote genomic stability [1]. The role of telomeric dysfunction in disease progression and cellular instability underscores the significance of maintaining telomeric health and the continuous attempts to develop telomerase-directed therapies [2]. Altered telomere length is associated with various pathologies.Accelerated telomeric shortening has been linked to the early onset of many age-associated ailments and to certain genetic disorders [3,4], including hematopoietic failure, some forms of ectodermal dysplasia, aplastic anemia, pulmonary fibrosis, liver disease, and premature biological aging [5]. Telomeric dysfunction has also been shown to be directly connected to cancer development.Individuals with shorter telomeres were found to be at increased risk of multiple tumors such as lung, bladder, and various gastrointestinal malignancies [6].Research has also shown that cancer cells often evade cellular apoptosis by employing mechanisms to counteract telomeric shortening; some do so by the activation of telomerase, leading to the addition of repetitive DNA sequences to chromosomal ends and sustaining telomeric elongation to enable cancer cells to undergo continuous divisions [6]. The POT1 protein is an essential subunit of the shelterin telomere binding complex.It directly binds to single-stranded telomeric DNA, protecting chromosomal ends from an inappropriate DNA damage response, and plays a role in telomere length regulation.Shelterin plays a vital role in telomere function by remodeling telomeric DNA into a protected structure and managing the regulation of telomere length.Loss or malfunction of shelterin proteins results in uncapped telomeres, which induce genomic instability.Lately, increasing evidence shows that long telomeres caused by variants in shelterin components (POT1, TPP1, and RAP1) also display an increased risk of cancer [7]. POT1 is involved in the regulation of telomerase-dependent elongation of the telomere.Some studies have suggested that it increases the processivity of telomerase, whereas others have identified a role of POT1 in the negative regulation of telomerase catalytic activity [8,9].POT1 is composed of three oligosaccharide/oligonucleotide (OB) fold domains, namely, OB1, OB2, and OB3.It binds to the telomeric single-stranded repeat via the OB1 and OB2 fold domains, while the OB3 fold domain enables its association with the TPP1 protein [10].Variants in the OB1 and OB2 domains affect the single-stranded DNA-binding ability of POT1, while variants in OB3 diminish POT1's affinity for TPP1, affecting its recruitment to telomeres [11]. Somatic POT1 variants were initially identified in CLL cells [12].Germline biallelic POT1 variants have been reported in an infant with Coats plus syndrome [13].Germline mono-allelic variants have been identified in various types of cancer in small series [12].Cutaneous melanoma is the most commonly reported malignancy in POT1 carriers.Previous publications have described POT1 variant carriers to be at increased risk of CLL, uterine cancer, lung cancer, sarcoma, and colon cancer.Functional studies have shown significant disruption of the protein in carriers [14][15][16].Most such reports were limited by the small number of carriers.Lately, POT1 variants associated with long telomere length were also shown to confer predisposition to a familial clonal hematopoiesis syndrome that is associated with a range of benign and malignant solid neoplasms [17]. The human POT1 gene is comprised of 22 exons.The (NM_015450.3) c.233T>C p.(Ile78Thr) variant is located in exon 7 within the OB1 domain.The variant has been submitted to ClinVar by several clinical testing laboratories (variation ID 475073) with conflicting interpretations of pathogenicity, including uncertain significance and likely pathogenic, supported by one star.A study published in 2019 by Wong et al. highlighted the potential pathogenicity of the p.(I78T) variant within families showing a predisposition to melanoma [18].In recent years, further research, functional assessments, and clinical data led to more submissions in ClinVar, claiming its pathogenicity and changing the variant classification to likely pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines. In this study, we review the previous allusions of the POT1 Ile78Thr variant, describe ten apparently unrelated carrier families, estimate its prevalence among AJs, and describe the clinical landscape among patients and families.This report could help to provide clinical data to design surveillance protocols and raise awareness to POT1 as a cancer predisposition gene.We advocate testing the p.(I78T) variant in high-risk patients of AJ descent and including POT1 in germline panels for various types of tumors. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "This cohort included eleven index patients found by genotyping to have the p.(I78T), (NM_015450.3)c.233T>C, chromosome 7, g.124870933A>G, (GRCh38) germline variant in the POT1 gene.Ten patients had been referred for oncogenetic counseling due to personal history of cancer; one was referred for trio-exome sequencing because of a developmental delay in her child.The relevant demographic, clinical, pathologic, and genetic data were retrieved from the medical files. Informed consent was obtained from all subjects involved in the study.The study was approved by the institutional review boards of all participating medical centers (approval no.0847-22). Testing was performed via one of three genetic institutes in Israel between 2018 and 2023.It consisted of either next-generation sequencing with multicancer panels (Invitae Multi-Cancer Panel, Invitae CORP, San Francisco, CA; Blueprint Comprehensive Hereditary Cancer Panel, Blueprint Genetics, Espoo, Finland; and Clalit Genomic Center Hereditary Cancer Panel, Clalit Genomic Center, Petach Tikva, Israel) and/or whole exome sequencing in the CeGat laboratory (CeGaT GmbH, Tübingen, Germany). The genomic DNA obtained from the submitted samples was enriched for the targeted regions using a hybridization-based protocol and sequenced using Illumina technology.Sequencing libraries were size-selected with the bead-based method to ensure optimal template size and amplified by polymerase chain reaction (PCR).Regions of interest (exons and intronic targets) were targeted using the hybridization-based target capture method.The resulting sequences were aligned against the human genome build GRCh37/UCSC hg19 and analyzed for sequence variants.The different laboratories used various methods for the identification of sequence changes (variants) in the aligned reads.All labs had processes in place for detecting copy number variations (CNV).The clinical interpretation of identified variants was based on established guidelines, considering databases such as gnomAD, ClinVar, and HGMD for variant assessment.Output files containing the sequenced data and variant information were produced.The full list of genes included, sequencing methods in each panel, and the genotyping technique of exome sequencing are explained thoroughly in Supplementary File S1. Haplotype analysis was completed by genetic testing for the microsatellite analysis.It was performed by using fluorescent PCR for size discrimination with capillary electrophoresis.Testing was performed on DNA extracted from the peripheral blood cells of six Ashkenazi individuals from different families.The PCR was performed in a 25 µL vessel containing 50% Taq Mix Red (PCR Biosystems Ltd., London, UK) and 5% dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MI, USA), using 0.1 µM of each primer.The reaction was thermocycled for 35 cycles using a program of 30 s at 72 • C. The reaction products were diluted, run on an ABI Prism 3100 Avant automated sequencer, and analyzed using GeneMapper software v6 (ABI).The primers for the polymorphic markers flanking the POT1 gene were TG1 (chr7:123,781,554), TG2 (chr7:124,091,551), AC1 (chr7:124,177,355), GT1 (chr7:124,189,252), GT2 (chr7:124,369,137), GT3 (chr7:124,428,420), GT5 (chr7:125,163,190), GT6 (chr7:125,288,073), AC2 (chr7:125,392,256), and AC3 (chr7:125,567,192).The forward primer for the variant was GGTTTGGTGTTTTGAAG-TAAGCA, and the reverse primer was TTTCTGGGGAATGAAAGCAG. ", "section_name": "Patients and Methods", "section_num": "2." }, { "section_content": "The p.(I78T) germline missense variant in the POT1 gene was detected in eleven probands (eight females, three males) from ten apparently unrelated families, all of AJ descent (Figure 1).Nine patients (82%) were diagnosed with melanoma between the ages of 25 and 63 years (Table 1).Seven of them (78%) reported recurrence of melanoma in different body parts, and most had undergone more than three resections at the time of the genetic consults.One patient (family 6, patient III-1) reported 10 recurrent resections, and one (family 4, patient 4-III-1) reported many additional resections of dermal nevi with high atypia.Tumor burden among carriers was high, with 30 primary malignancies (range 1-6; ages 25-67) (Table 1).Eight index patients reported a wide array of tumors.They included breast cancer in five (63% of all female carriers; ages 44, 44, 54, 51, and 67), sarcoma in two, desmoid tumors in three, and PTC in three.Other tumors included CLL, oligodendroglioma, neuroendocrine tumor, pheochromocytoma, lung cancer, urinary transitional cell carcinoma (TCC), colorectal cancer, prostate cancer, and adenomatous polyposis.One patient (family 10, patient 10-II-1), who was a heavy smoker, was first diagnosed with cancer at age 59 and had six different tumors, including multi-focal nonsmall-cell lung adenocarcinoma and TCC.The age range for the occurrence of the first tumor was 25-59 years.For two of them, melanoma was not the first tumor to occur.Abbreviations: CLL: chronic lymphocytic leukemia, PTC: papillary thyroid cancer.Family history of various malignancies was reported in nine families (90%) (Figure 1, Table 1).The proband of family 1 reported two daughters diagnosed with malignant melanoma at ages 20 and 27 (patients III-1; III-3); one of them (patient III-1) also had a desmoid tumor at age 40.The proband of family 4 reported two relatives with CLL (patients II-2 and III-2), and the proband of family 6 reported three relatives with earlyonset melanoma (patients I-2, II-1, and II-2).Proband III-1 from family 8 had a child who died from pediatric neuroblastoma.In six families (1, 3, 4, 6, 8, and 9) the age of first cancer reported was earlier in the younger generation; however, genotyping was only available for probands and not for the affected family members.Cascade testing was performed on two healthy carriers from family 8 (patients IV-2 and IV-3) who were found to be carriers for the familial variant at the age of 18 and 21 and were referred to clinical evaluation. Other genes associated with the common tumors reported, especially melanoma, and the common malignancies of colorectal, breast, and prostate cancer, were included in all tests performed (Supplementary File S1).Only one patient (family 6, patient III-1) was found to carry an additional disease-causing variant, c.1273G>A p.Glu425Lys, in the MITF gene (NM_001354604.2),also known to be associated with melanoma.This patient reported a high recurrence rate of melanoma, having had 10 different resections over the years. To gauge the allelic frequency of the p.(I78T) variant in the AJ population, a review of a local exome database was conducted.The database comprised 2721 exomes analyzed from 2018 to 2023 for various morbidities, mainly intellectual disability.The allelic frequency of the p.(I78T) variant across the whole database was 0.06% and was 0.25% among individuals identifying themselves as AJs. Haplotype analysis was conducted in six of the carriers of unrelated families.The 10 microsatellite markers that were tested represent the haplotype and are presented in Supplementary File S1.All the carriers tested were found to share the same haplotype. ", "section_name": "Results", "section_num": "3." }, { "section_content": "The (NM_015450.3) c.233T>C p.(Ile78Thr) variant is located in a conserved domain of POT1 that interacts with telomere DNA.Functional studies have shown that it alters the OB1 region, leading to the impairment of POT1 single-stranded DNA binding and resulting in increased telomere length [11].Assays of POT1 proteins and mRNA in lymphoblastoid cell lines derived from carriers of POT1 p.(I78T) showed decreased expression and defective binding to telomere DNA, indicating that this missense variant has a loss-of-function effect.It has been shown that haploinsufficiency for POT1 facilitates telomerase-dependent telomere elongation, and that long telomeres extend the replicative potential of cultured cells [11].Telomere elongation is associated with telomere fragility, replication defects, and genomic instability.Indeed, carriers of POT1 pathogenic variants were shown to have longer telomeres, and progenitors from POT1 carriers displayed elevated somatic mutation burden compared to noncarriers [17]. Conflicting interpretations of pathogenicity are reported in ClinVar (variation ID# 475073); however, the functional support and the multiple prediction scores (e.g., SIFT = 0, REVEL = 0.69, and alphamissense 0.7842) suggest that it is deleterious.Bioinformatic analysis according to the guidelines for the classification of sequence variants of the ACMG [19] was carried out.PS4 criteria were used according to previously described patients and those described in our work, as elaborated in Table 2; PS3 criteria were used based on functional studies that have shown that the variant disrupts POT1-telomere binding and results in elongated telomeres as compared to controls [17].PM1 criteria were used as the variant is located in the POT1 domain, with a missense constraint value of 0.49, and other reported missense variants.For PM2, the variant frequency in the gnomAD v4 database is 0.0087%, while in the AJ sub-population the frequency is 0.02%.However, PM2 criteria can still be used, following the ACMG recommendations of downgrading it to a supporting level; PP3 criteria were used as computational prediction tools support a deleterious effect on the protein (REVEL 0.69, alphamissense 0.784).Taken together, based on the scoring of PS4 (4), PS3_moderate (2), PM1 (2), PM2_supporting (1), and PP3(1), the variant was classified as pathogenic.This variant has been mentioned previously (Table 2).All five previous publications describe the high occurrence of melanoma, and three report lymphoid malignancies among carriers; two have also described the occurrence of thyroid malignancy.The current publication describes a wider spectrum of solid malignancies apart from melanoma; we describe breast cancer in five of the eight female carriers, abdominal desmoid tumors in three carriers, colonic polyps in two carriers, and other tumors (Table 2). Carriers in our cohort had high risk of recurrent melanoma (3 to 10), starting at the age of 25, earlier than the previously reported age of 31 [18].In one first-degree non-genotyped family member, a melanoma had already appeared already at 20 years old.Interestingly, the sole patient found to be a \"double carrier\" of the POT1 and MITF variants had an extremely high recurrence rate of melanoma (reporting 10 different resections over the years), possibly reflecting an additive/synergistic effect of the two variants. CLL was diagnosed in one carrier and in two family members of another carrier.POT1 was previously reported to be a candidate gene in lymphoid malignancies.No other genes are known to be associated with a definite risk of CLL.A recent study of 576 patients with CLL found a 3.3% prevalence rate of disease-causing germline and somatic loss-of-function variants in POT1.Others reported the poor prognostic implications of somatic and germline POT1 variants in carriers with CLL, including shorter survival times and reduced response to treatments [23,24].Desmoid tumors were reported in four families in our cohort, two located intraabdominally and two within the abdominal wall.Tumors were recurrent and locally aggressive in all cases, appearing in different areas and requiring surgery.Disease onset occurred at ages 40, 41, 60, and 66.None of the patients had had previous abdominal trauma or surgery, both risk factors for abdominal desmoids. Three of the patients with desmoid tumors underwent genetic testing and were found to carry the POT1 variant.It is of note that one of these three carriers also had multiple adenomatous colonic polyps, raising the suspicion of APC-associated polyposis; however, neither him, nor the other two patients with desmoids, were found to carry a diseasecausing variant in APC or in other polyposis-related genes. Abdominal desmoids are rare fibroblastic tumors.They usually do not metastasize, but they are locally aggressive.Most are not associated with inherited conditions.The only gene reported so far to be associated with a definite risk for desmoids is APC (5-15%).In a small series of patients with desmoid tumors, disease-causing variants were identified in CHEK2, ERCC5, BLM, MSH6, and PALB2 [25]; however, none of these genes has been shown to be associated with increased risk of having desmoids.In the present study, desmoid tumors were reported in 40% of the evaluated families; a mesenteric desmoid tumor was reported recently in one POT1 carrier [17].To the best of our knowledge, these are the first reports of desmoid tumors among carriers of a disease-causing POT1 variant. Three carriers (family 5, family 9, and family 10) were diagnosed with recurrent PTC at ages of 31, 41, and 63 years and were operated on more than once.Recently, POT1 was suggested to be a candidate gene for familial non-medullary thyroid cancer based on the observation of a single family in which multiple members diagnosed with PTC were found to be carriers of the p.(V29L) variant in POT1 [26].None of these carriers were reported to have melanoma, CLL, or desmoid tumors.PTC is reported in two other families with the p.(I78T) variant (Table 2).The association between POT1 and thyroid cancer remains provisional and justifies further research. DeBoy et al. [17] suggested occurrence of genetic anticipation, wherein offsprings of POT1 disease-causing variant carriers had cancer that developed several decades earlier than that in their parents.Our data also suggest anticipation, based on the age of occurrence of the first tumor in families 1, 3, 4, 6, and 8.The proband of family 1 was diagnosed with melanoma at the age of 40, while her two daughters developed the disease at ages of 20 and 27; the proband of family 6 developed melanoma 10 years earlier than her mother and uncle; and the proband of family 8 had a child with neuroblastoma.However, as most family members have not been genotyped, this aspect should be further studied. We did not look into the pathological or molecular features of the tumors reported.One study reported the common occurrence of spitzoid melanocytic neoplasms, characterized by large epithelioid melanocytes with abundant cytoplasm in POT1 variant carriers, where 23 of the 30 melanomas studied had a spitzoid morphology involving at least 25% of the tumor [21].Looking for unique features of POT1-associated tumors may provide insights regarding tumor behavior and, perhaps, clues for diagnosis. All carriers in our study were AJs.The variant is very rare among healthy individuals, with allelic frequency in the gnomAD v.4.0.0 dataset of 0.0009%, detected in individuals from Ashkenazi and European descent.No homozygotes have been reported.Haplotype analysis revealed a shared haplotype, indicating that a common founder was likely in these families.We deduce that the carrier rate in AJs might be higher in patients with melanoma, particularly with early age of onset, more than one malignancy or significant family history. There are no published guidelines for the surveillance of individuals with a POT1 tumor predisposition.It has been suggested that decisions regarding individual surveillance protocols should be based on the emerging phenotype spectrum of POT1 variants and on the affected individual's personal and family history.In addition, it has been recommended to employ screening similar to that used in LFS, due to the similarity to Li-Fraumeni syndrome [27].In line with this suggestion, we recommend that carriers of POT1 variants be referred for dermatological examination at least every 6 months beginning at the age of 18 years.It may be prudent to also refer carriers of the p.(I78T) variant to surveillance for thyroid, breast, and colon cancer, or to consider whole body MRI.On the basis of the present series, we advocate that individual surveillance protocols should be determined with consideration of both the evolving phenotypic spectrum and the personal and family history of the affected individuals and also take into consideration the variant involved. ", "section_name": "Discussion", "section_num": "4." }, { "section_content": "Here, we describe patients from ten unrelated families of AJ descent found to carry a disease-causing missense variant in the POT1 gene, previously classified as VOUS.This is a founder variant with allelic frequency among AJs of 0.25%.This variant is associated with high tumor burden, risk for melanoma of earlier onset than previously reported, and an unprecedented link with various solid tumors, especially desmoid tumors, PTC, and breast cancer.This missense variant affects the OB1 region of the protein, resulting in increased telomeric length.We advocate testing for this variant in high-risk patients of Ashkenazi descent.In addition, the inclusion of POT1 in germline panels for various types of cancer is warranted.Further research is needed to define the landscape of cancer types, establishing guidelines for surveillance, and deciphering the molecular characteristics of the resulting tumors and their possible varying responses to treatments. ", "section_name": "Conclusions", "section_num": "5." } ]	[ { "section_content": "The data presented in this study are available on request from the corresponding author.The data are not publicly available due to privacy and ethical issues. ", "section_name": "Data Availability Statement:", "section_num": null }, { "section_content": "The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes15030355/s1,File S1: The list of tested genes, panel's sequencing methods, and the genotyping technique of exome sequencing.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. The authors declare no conflicts of interest. ", "section_name": "Supplementary Materials:", "section_num": null }, { "section_content": "The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes15030355/s1,File S1: The list of tested genes, panel's sequencing methods, and the genotyping technique of exome sequencing.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. ", "section_name": "Supplementary Materials:", "section_num": null }, { "section_content": "The authors declare no conflicts of interest. ", "section_name": "Conflicts of Interest:", "section_num": null }, { "section_content": "", "section_name": "Abbreviations", "section_num": null } ]
10.1038/s41419-024-07172-w	RAB4A is a master regulator of cancer cell stemness upstream of NUMB–NOTCH signaling	<jats:title>Abstract</jats:title><jats:p>Cancer stem cells (CSCs) are a group of specially programmed tumor cells that possess the characteristics of perpetual cell renewal, increased invasiveness, and often, drug resistance. Hence, eliminating CSCs is a major challenge for cancer treatment. Understanding the cellular programs that maintain CSCs, and identifying the critical regulators for such programs, are major undertakings in both basic and translational cancer research. Recently, we have reported that RAB4A is a major regulator of epithelial-to-mesenchymal transition (EMT) and it does so mainly through regulating the activation of RAC1 GTPase. In the current study, we have delineated a new signaling circuitry through which RAB4A transmits its control of cancer stemness. Using in vitro and in vivo studies, we show that RAB4A, as the upstream regulator, relays signal stepwise to NUMB, NOTCH1, RAC1, and then SOX2 to control the self-renewal property of multiple cancer cells of diverse tissue origins. Knockdown of NUMB, or overexpression of NICD (the active fragment NOTCH1) or SOX2, rescued the in vitro sphere-forming and in vivo tumor-forming abilities that were lost upon RAB4A knockdown. Furthermore, we discovered that the chain of control is mostly through transcriptional regulation at every step of the pathway. The discovery of the novel signaling axis of RAB4A–NUMB–NOTCH–SOX2 opens the path for further expansion of the signaling chain and for the identification of new regulators and interacting proteins important for CSC functions, which can be explored to develop new and effective therapies.</jats:p>	[ { "section_content": "Cancer stem cells (CSCs) possess a self-renewal capacity that is important not only for tumor initiation, maintenance, and progression, but also enhanced resistance to many modalities of therapies, contributing to treatment failure and cancer recurrence [1,2].Epithelial-mesenchymal-transition (EMT), a biological process where epithelial cells acquire mesenchymal-like characteristics, is associated with the acquisition of stem cell-like properties and tumor progression [3,4].Due to their central roles in cancer, targeting EMT and cancer stemness has emerged as a promising strategy to improve cancer treatment outcomes [5].To this end, identifying the factors that are important for EMT and cancer stemness can pave the way for the development of targeted therapies aimed at disrupting EMT and eliminating CSCs to improve clinical outcomes. NUMB is a protein that plays a crucial role in the regulation of cell fate, including its involvement in CSCs.During development, NUMB is a determinant in the asymmetric division of stem cells to maintain tissue homeostasis; in the cancer cell, it acts mostly as a tumor suppressor in reducing self-renewal capacity and stemness thus limiting tumor growth [6,7].Though a known tumor suppressor, NUMB has context-dependent roles in specific cancers highlighting the complexity of its functions and emphasizing the importance of a clear understanding of the signaling circuitry [8][9][10][11][12]. NOTCH protein signaling is also highly conserved in various cellular processes, including development, differentiation, and tissue homeostasis.In recent years, the pro-cancer roles of NOTCH signaling in cancer stemness and drug resistance have garnered significant attention [13][14][15].NOTCH signaling cross-talks with several other pathways involved in drug resistance, such as PI3K/ Akt and MAPK/ERK.As such, therapeutic targeting of NOTCH and its downstream effectors has shown promise in preclinical and clinical settings [16].As with many other proteins that play roles in cancer, the mechanism of regulation by NOTCH is also cell context-dependent [17][18][19][20][21]. SOX2 (sex-determining region Y-box 2) is one of the key transcription factors involved in maintaining the pluripotency and self-renewal capacity of embryonic stem cells; it is one of the four so-called Yamanaka factors that induce pluripotent stem cells [22].In cancer, SOX2 expression has been detected in CSC populations across various tumor types [23,24].It interacts with other transcription factors and signaling pathways to orchestrate the balance between self-renewal and differentiation of CSCs.As such, SOX2 is known to induce EMT and mediate drug resistance [25][26][27][28].Therefore, targeting SOX2 can potentially reduce cancer cell self-renewal capacity and enhance the effectiveness of cancer treatments; however, little progress has been made due to the difficulty in directly targeting transcription factors [27]. A common theme has emerged that the roles of the aforementioned cancer stem cell regulators are complex and cell context-dependent in biology and in cancer, which underscores the importance of a better understanding of the regulatory mechanisms.Recently, we reported the RAB4A-RAC1 axis of signaling as a major regulator for EMT and cancer stemness [29].RAC1, a member of the Rho family of small GTPases, is known for its roles in regulating cellular processes such as cytoskeletal dynamics, cell migration, and gene expression [30,31].Activation of RAC1 is also reported to contribute to the evasion of cell death induced by chemotherapy or targeted therapies, leading to resistance and tumor recurrence [32][33][34][35][36][37][38][39].In addition, the crosstalk of RAC1 with many signaling pathways, such as those of Wnt/ β-catenin, PI3K, and MAPK, is important in tumorigenesis [34,35]. In the previous study, we found RAB4A to be a major upstream regulator of RAC1 activation on EMT and cancer stemness [29].However, there are gaps/missing signaling components in the transmission of this regulation that need to be identified.In this paper, we report a novel signaling cascade of RAB4A-NUMB-NOTCH-RAC1-Sox2 as a major and fundamental driver in promoting cancer stemness and tumorigenesis, significantly extending the mechanistic understanding of the role of RAB4A-RAC1 regulation of EMT and stemness signaling. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "Human breast cancer cell lines MDA-MB-231, MCF7, human prostate cancer cells PC3, and human glioblastoma cells SNB19 were obtained from the American Type Culture Collection (ATCC) (Rockville, MD) and were tested mycoplasma negative.These cells were cultured in Dulbecco's minimal essential medium (DMEM, Nacalai, California, USA) supplemented with 10% v/v FBS and penicillin (100 U/mL)/streptomycin (100 μg/mL) from Hyclone (IL, USA) following ATCC standard conditions.The actinomycin D (ThermoFisher Scientific, USA) treatment of cells was conducted using the established protocols [40]. ", "section_name": "MATERIALS AND METHODS Cell culture", "section_num": null }, { "section_content": "HEK293T cells were used to make the viruses for stable knockdown and stable protein over-expression.Transfection was done by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) using standard lab protocols [41].For transduction in the cell line of interest, the virus-containing-media from HEK293T cells was mixed with fresh 10% FBS DMEM at 50% v/v and with a final concentration of 8 μg/mL polybrene (#H9268, Sigma-Aldrich).The stable knockdown of RAB4A and NUMB was achieved using the shRNA plasmids obtained from Sigma-Aldrich, USA (Supplementary Table S1A).RAB4A overexpressing cells were generated as described previously [42].NICD and SOX2 genes were amplified from the cDNA obtained from MDA-MB-231 cells (Supplementary Table S1B).RAC1 expressing constructs were generated as described previously [29].RAB5A siRNA (#EHU053901, Sigma-Aldrich, USA) transient transfections were performed using Lipofectamine 3000 reagent (ThermoFisher Scientific, USA) and associated manufacturer's protocol. ", "section_name": "Generation of stable cell lines and transient transfection", "section_num": null }, { "section_content": "Total RNA extraction and cDNA conversion were done using Tissue Total RNA Mini Kit (FATRK 001-2, Favorgen Biotech Corporation) and ReverTra Ace qPCR RT Master Mix (FSQ-201, Toyobo).All qPCR measurements were performed using the BIO-RAD CFX96 (California, USA) and SYBR green (#4913850001, Roche, WI).The primers used for qPCR are listed in Supplementary Table S1C.Cancer stemness gene expression analysis was done according to the manufacturer's protocol using the predesigned qPCR multiplex array \"Cancer stem cells (SAB target list) H96, Bio-Rad, USA\".The heat map was generated using online Morpheus analysis software. ", "section_name": "Quantitative PCR (qPCR)", "section_num": null }, { "section_content": "Nuclear extraction was performed using the protocol from Thermofisher for cellular fractionation (https://www.thermofisher.com/sg/en/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/elisa-sample-pre paration-protocols/nuclear-extraction-method-.html).Briefly, 5 × 10 6 cells were collected in ice-cold PBS, lysed using the hypotonic buffer and 10% NP-40 and cytoplasmic fraction was collected following the centrifugation step.The remaining nuclear pellet was further treated with the cell extraction buffer and used for analysis.For the whole cell immunoblotting, Cells were lysed with RIPA buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.1% SDS) containing protease and phosphatase inhibitors (#4693159001, Roche, WI, #P0044, Sigma-Aldrich, St. Louis, MO), and processed using lab standard protocol [43].RAC1-GTP pulldown and analysis were performed using a kit from Cytoskeleton, Inc. (BK035; Denver, CO) according to the manufacturer's protocol.The antibodies used are listed in Supplementary Table S1D. ", "section_name": "Cellular fractionation, RAC1 pull-down assay, and immunoblotting", "section_num": null }, { "section_content": "A 2562 base pair fragment upstream of NUMB gene with enriched binding of RNA Polymerase II and H3K4me3 was identified using Cistrome data and UCSC genome browser (http://cistrome.org/db/#/) in MDA-MB-231 and MCF7.The promoter of NOTCH1 was identified for MDA-MB-231 cells [44].The NUMB and NOTCH1 promoter regions were amplified from MDA-MB-231 genomic DNA (Supplementary Table S1E) and sub-cloned into a pGL3-Basic vector (Promega, Madison, WI).MDA-MB-231 and MCF7 (4 × 10 5 cells) cells with stable RAB4A knockdown or RAB4A overexpression were transiently transfected with either pGL3 control vector or pGL3 expressing NUMB and NOTCH promoter regions using the Lipofectamine 2000 protocol (ThermoFisher Scientific, USA).A vector expressing Renilla was co-transfected as a control for the normalization of luciferase activity.Luciferase was measured after 24 h of transfection using the Dual-Luciferase Reporter Assay System (#E1960, Promega Pte Ltd) as per the manufacturer's protocol. ", "section_name": "Luciferase assay", "section_num": null }, { "section_content": "Sphere formation was used to study the cancer stemness and was performed as described previously [45].Briefly, cells were seeded at 400 cells/well in DMEM-F12 containing 0.5% methylcellulose (Sigma-Aldrich, MO, USA), B-27, and N2 (Gibco, MD, USA) in low-adherent culture plates (#3474, Corning) and were cultured for two weeks.For serial plating, spheres were treated with StemPro® Accutase® Cell Dissociation Reagent (Gibco) resuspended, and seeded as mentioned above.Sphere count was analyzed using Open CFU software (Geissmann). ", "section_name": "Tumor sphere formation assay", "section_num": null }, { "section_content": "All animals were treated in accordance with the IACUC Guidelines (protocol no.2021/SHS/1627).All mice were housed in a temperature and light-controlled environment (12 h light, 12 h dark) and were provided food and water ad libitum.For all the cell lines tested, 5 × 10 6 cells, harvested in DMEM containing 10% FBS and 50% Matrigel (BD sciences), were injected subcutaneously into the flank of 8-week-old NOD-SCID-Gamma female mice.Although randomization was not employed in this study, efforts were made to minimize bias by including at least 5 mice per group.When the tumors reached 1 cm 3 (L × W 2 /2), the mice were euthanized by CO 2 inhalation followed by cervical dislocation, and the tumors were removed and processed. ", "section_name": "Animals and xenografts", "section_num": null }, { "section_content": "Statistical analysis was carried out using GraphPad Prism software (GraphPad, CA, USA).Data are presented as the mean ± standard error of the mean (SEM) and represent at least three independent biological replicates.Statistical significance was determined by either Student's unpaired t-test, one-way ANOVA, or two-way ANOVA.ANOVAs were followed with Dunnett's multiple comparisons or Tukey's post hoc test.p < 0.05 is considered significant. ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "In our recent studies, we found that RAB4A is essential for the continuous proliferation and maintenance of EMT features in cancer cells of multiple tissue origins [29].We also discovered that this role of RAB4A acts through controlling RAC1 activation. RAB4A suppression leads to the loss of cancer cell sphere formation in vitro and loss-of-tumor formation in both subcutaneous and orthotopic xenograft mouse models (Fig. 1A, B and Supplementary Fig. S1) [29]; this loss of tumorigenesis can be rescued by exogenous expression of constitutively active RAC1 (RAC1 CA ) (Fig. 1A, B and Supplementary Fig. S1) [29].Table 1 summarizes the results of the two mouse-model studies.A Representative photos of tumors for each group at the endpoint of the experiment; the tumors are derived from MDA-MB-231 cells that express either control shRNA or that target RAB4A with and without concurrent exogenous RAC1-CA expression.B Tumor formation and growth for each group as described in (A).Prism software was used for the analysis.The group identities and numbers of mice in each group are written in the inset of the figure.C-G qPCR analysis was performed using prime PCR multiplex assay that contains 87 genes that have been validated for their roles in cancer stemness.Relative mRNA expression was calculated compared to the control cells.Genes that were altered consistently by the two shRNA sequences targeting RAB4A are represented.The heat map was generated using the Morpheus software.The fifth column denotes the direction of the regulation in a typical cancer stemness phenotype based on published research [54][55][56][57][58][59][60][61][62][63][64][65][66][67][68].C Group scatter plot for the 28 genes that are consistently altered by RAB4A-shRNA#1 and #2.D-G Heat maps of the gene set that are changed by RAB4A and rescued by RAC1 to different levels: D to the base levels, E above the base level, F below the base level, and G not rescued by RAC1. The effect of RAB4A KD on tumor formation is likely from RAB4A function on stem cell signaling as we have recently found that RAB4A affects EMT.To provide more evidence, we have performed some relevant experiments to rule out the potential effects of RAB4A on proliferation and cell death, which can also contribute to tumor formation.To this end, we observed no effect of RAB4A on cell proliferation nor apoptosis assayed on adherent cultures (Supplementary Fig. S2A,B).Serial-replating sphere formation assay is a commonly used assay to distinguish the short-term effect on proliferation or viability from that on the long-term self-renewal ability of the cells under study.The initial plating, i.e., firstgeneration sphere formation can be an indicator of the shortterm or acute effect on the cells; however, the subsequent replating sphere formation assesses the long-term self-renewal or stemness of the cancer cells.From the serial-replating sphere formation assays on the effect of RAB4A KD, the sphere numbers between control cells and the RAB4A KD cells are distinctly different at third replating (gen-3) but not significantly different at the initial plating (gen-1) (Supplementary Fig. S2C), consistent with the results from viability and proliferation study to support the conclusion that the impact of RAB4A KD is mainly on long term self-renewal ability, i.e., stemness. To delineate the molecular mechanism(s) accountable for the strong RAB4A-RAC1 signaling in the regulation of stemness and tumorigenesis, we performed a differential gene expression analysis on a group of experimentally validated CSC-related genes (SAB target list H96, Bio-Rad).Specifically, the expression levels of these stem genes were compared among MDA-MB-231 control cells, those stably expressing RAB4A shRNA, and those concurrently expressing RAB4A shRNA and CA-RAC1.Two RAB4A targeting shRNAs were used to assess the effects of RAB4A knockdown, and for each shRNA, CA-RAC1 was introduced to assess the rescue effects.The stable RAB4A knockdown and RAC1 activation status in these cell lines were validated before the geneexpression analysis (Supplementary Fig. S3).Among the 87 genes tested, the expression of 27 showed consistent changes between the RAB4A-shRNA1 and RAB4A-shRNA2 expressing cells and between the two sets of CA-RAC1 rescue cells (Supplementary Table S2 and Supplementary Fig. S4).Strikingly, almost all the genes that were consistently changed were down-regulated upon RAB4A knockdown, demonstrating the central role of RAB4A in maintaining stemness (Fig. 1C).For a large proportion of the down-regulated genes, the expression was reversed by CA-RAC1 expression (Fig. 1D).Further, given that the gene expression changes are under the same RAB4A knockdown and CA-RAC1 expression, the level of CA-RAC1 rescue for each gene provides clues on the extent of RAC1 involvement downstream of RAB4A in regulating stemness/EMT.For the first group of genes, CA-RAC1 expression reverses the expression to near the baseline level, suggesting a linear RAB4A-RAC1 axis as the sole regulation (Fig. 1D).For the second group of genes, CA-RAC1 expression overcompensated the RAB4A knockdown effect, demonstrating a broader and bigger role of RAC1 than that of RAB4A in regulating the expression of the gene (Fig. 1E).For the third group of genes, the CA-RAC1 expression only partially reversed the effect of RAB4A knockdown, suggesting that downstream of RAB4A there may be other regulator(s) besides RAC1 (Fig. 1F).For yet another group, CA-RAC1 expression makes no difference to the RAB4A regulated expression, which implies two possibilities-either these genes are upstream of RAC1 in RAB4A signaling or RAB4A signals through pathway(s) independent of the said gene(s) (Fig. 1G). In summary, the expression analysis of the select panel of CSCrelated genes showed that RAB4A is a master regulator of CSCs and EMT, and that RAC1 is the major downstream mediator for much of this role of RAB4A (Fig. 1C-F).Even for the group of genes that are not rescued by CA-RAC1 expression, they could be upstream of RAC1 in the RAB4A signaling especially considering that they are mostly cell surface proteins. ", "section_name": "Gene expression analysis reveals that RAB4A regulates transcription program(s) controlling cancer stemness through the activation of RAC1", "section_num": null }, { "section_content": "The differential gene expression of CSC regulators in response to RAB4A knockdown and CA-RAC1 expression (Fig. 1) is consistent with the tumorigenesis phenotypes associated with RAB4A and RAC1 functions that we have shown in both previous [29] and current in vitro and in vivo studies (Fig. 1 and Table 1).As the pathway signatures identified RAB4A as a major upstream stem cell regulator, we next explored the changes in the levels of major cancer-driving proteins in MDA-MB-231 control and RAB4A knockdown cells.Among these cancer-signaling proteins, we observed a significant reduction of NOTCH1 and SOX2 levels and, in contrast, a dramatic elevation of NUMB protein levels upon RAB4A knockdown (Supplementary Fig. S5); the NOTCH1 and SOX2 changes are consistent with the gene expression results as presented in Fig. 1.To facilitate the studies on the impact of RAB4A, we assayed a group of cancer cell lines for the expression of RAB4A and categorized them into RAB4A-high and RAB4A-low groups (Fig. 2A).We then validated the levels of NUMB, NOTCH, and SOX2 in a number of RAB4A-high cancer cells (MDA-MB-231, PC3, and SNB19) in response to RAB4A knockdown (Fig. 2B), and in RAB4A-low cancer cells (MCF7 and H1299) in response to RAB4A over-expression (Fig. 2C).The findings confirmed that RAB4A positively controls levels of NOTCH1 and SOX2, and negatively of NUMB, in these cancer cells.We then looked at the transcript levels of these genes and found that, consistent with the protein levels, the transcription of Notch1, Sox2, and the stem cell marker Aldh1a3 genes were suppressed, while that of Numb was elevated, in response to RAB4A knockdown in RAB4A-high cancer cells (Fig. 2D-F).In the RAB4A-low cells, over-expression of wildtype RAB4A, but not the inactive mutant protein RAB4A(S27N) nor the C-terminal deletion form RAB4A(ΔCXC), increased the expression of Notch1, Sox2, and Aldh1a3 while reducing Numb expression (Fig. 2G,H).These observations on both protein and transcript levels suggest that RAB4A controls these cancer stemness regulators mainly through gene expression.Constitutively active RAC1 expression reverses the RAB4A knockdown effect on SOX2 but not NUMB or NOTCH1 expression From our recent report and the above stemness gene expression analysis, we know that RAC1 is a major mediator for RAB4A regulation of EMT and cancer stemness.With the validation in multiple cell lines that RAB4A controls the transcription of Numb, Notch1, and Sox2 genes, we proceeded to study the role of RAC1 on these genes.For this assessment, we performed the protein quantification on nuclear and cytosolic fractions since NUMB, NOTCH1, and SOX2 all have major functions in regulating gene transcription and their cellular localization impacts these functions.First, we found that, in the RAB4A-high MDA-MB-231 breast cancer cells, NUMB expression is essentially absent and NOTCH1 and SOX2 expression are easily visualized by western blot (Fig. 3A).NICD, the active NOTCH1 fragment, and SOX2 are exclusively localized in the nucleus while the full-length NOTCH1 is mostly in -------- -----+ ------RAB4A KD#1 -+ + + ----+ + + ---RAB4A KD#2 ----+ + + ----+ + + OE WT-RAC1 --+ --+ ---+ --+ -OE CA-RAC1 ---+ --+ ---+ -- Cell lysates and nuclear/cytosol fractionation were prepared for the western blots. ", "section_name": "RAB4A regulates the expression of critical stem cell factors NUMB, NOTCH, and SOX2", "section_num": null }, { "section_content": "the cytosolic fraction as expected (Fig. 3A).Distinct from the impact of RAB4A knockdown, RAC1 knockdown only abolished the expression of SOX2, but caused no noticeable change in NOTCH1/NICD and NUMB levels (Fig. 3A).We then evaluated the rescue effect of RAC1 over-expression in RAB4A knockdown cells on the levels of these stem cell factors.As presented earlier, RAB4A knockdown completely abolished the NOTCH1/NICD expression and induced NUMB expression that is exclusively localized in the nucleus (Fig. 3B).Notably, neither NOTCH nor -----------+ Ctrl KD + --+ --NUMB KD#1 -+ --+ -NUMB KD#2 --+ --+ GTPgS + --+ --+ --GDP -+ --+ --+rec-RAC1 --------- expression (Fig. 3B).In contrast, the expression of SOX2, lost from RAB4A knockdown was restored by CA-RAC1 but not WT-RAC1 (Fig. 3B).Consistently, pulldown assays using p21 binding domain (PBD, which pulls down only the activated RAC1/GTP-bound RAC1) confirmed that only the expression of CA-RAC1 but not WT-RAC1 increases the level of GTP-bound RAC1 in MDA-MB-231 cells (Fig. 3C). ", "section_name": "GTPgS + --+ --+ --+ --GDP -+ --+ --+ --+rec-RAC1 ------------+ GTPgS + --+ --+ --GDP -+ --+ --+rec-RAC1 -", "section_num": null }, { "section_content": "The impact of RAB4A and RAC1 was also evaluated in the RAB4Alow breast cancer cell line MCF7.In this case, we observed high baseline NUMB and low baseline NOTCH and SOX2 protein levels (Fig. 3D), opposite to those in the RAB4A-high cells.When WT-RAB4A was expressed, the NUMB level was suppressed while the NOTCH1 and SOX2 levels were induced; these effects of WT-RAB4A were obliterated by RAC1 knockdown (Fig. 3D).The effects on NUMB, NOTCH1 and SOX2 by WT-RAB4A were not observed by the expression of either RAB4A(S27N) or RAB4A(ΔCXC) (Fig. 3D), consistent with the notion that the C-terminal lipid modification site and the activation status of RAB4A are necessary for its regulation of stemness. Following the RAB4A expression study, we also assessed the effects of introducing RAC1 in MCF7 cells.In this case, we compared the control cells (empty expression vector) with those cells expressing either WT-RAC1 or CA-RAC1 proteins.Validating with the PBD pulldown RAC1 activity assay, we found that there is little activation of RAC1 in MCF7 cells at baseline, in contrast to the high activation level in MDA-MB-231 cells, despite the presence of similar levels of total RAC1 protein (Fig. 3E).This observation is consistent with the role of RAB4A in regulating RAC1 activation [42].The pulldown assay also confirmed that expression of CA-RAC1 dramatically increased the level of activated RAC1 while WT-RAC1 contributes little to the activity of RAC1 (Fig. 3E).CA-RAC1 had no effect on NUMB and NOTCH1 protein levels while caused significant increase in SOX2 level (Fig. 3F), consistent with the hypothesis that RAC1 is upstream of SOX2 in the RAB4A signaling chain.Notably, WT-RAC1 increases SOX2 at a much-attenuated level compared to CA-RAC1 (Fig. 3F), consistent with the conclusion from MDA-MB-231 cells (Fig. 3B) that the activation is required for RAC1 to increase breast cancer cell stemness.In conclusion, these studies in both RAB4A-high and RAB4A-low cancer cells demonstrate that RAC1 is upstream of SOX2 in the RAB4A regulation of cancer stemness, while the order of regulation among RAC1, NUMB, and NOTCH1 are yet to be defined. ", "section_name": "GTPgS + --+ --+ --+ --GDP -+ --+ --+ --+rec-RAC1 -", "section_num": null }, { "section_content": "We have presented evidence that links NUMB to RAB4A regulation for cancer cell stemness (Figs. 2 and3), and that loss-of-RAB4A leads to an increased expression of NUMB protein in RAB4A-high cancer cells and exogenous expression of RAB4A in RAB4A-low cells suppresses NUMB protein (Fig. 3).We also found that RAC1 regulates the expression of SOX2 but not NUMB or NOTCH1.To further delineate this signaling pathway, the role of NUMB regulation in the RAB4A-RAC1 axis of signaling was evaluated.To this end, we assessed the effect of the simultaneous knockdown of RAB4A and NUMB in MDA-MB-231 cells.Here we observed that, by knocking down NUMB, the effects of RAB4A knockdown on the suppression of NOTCH1 and SOX2 expression were reversed, suggesting that NUMB is upstream of these two proteins in the RAB4A regulation of stemness (Fig. 4A).We also found, in this setting, that RAC1 activity was restored with NUMB knockdown (Fig. 4B).Phenotypically, supressing NUMB restored the serial replating sphere formation ability that was lost due to RAB4A knockdown (Fig. 4C), demonstrating the essential role of NUMB in RAB4A regulation of self-renewal. The relevance of NUMB is also evaluated in the RAB4A-low, NUMB-high MCF7 breast cancer cells.Here, the knockdown of NUMB increased NICD/NOTCH1 and SOX2 protein levels, with SOX2 and NICD mostly in the nuclear fraction, consistent with the findings from MDA-MB-231 cells (Fig. 4D).RAC1 activity assay shows that knockdown NUMB increased GTP-bound RAC1 level, consistent with the notion that NUMB as an upstream regulator of RAC1 (Fig. 4E).Serial replating sphere formation assay showed that suppressing NUMB dramatically increased the self-renewal ability of MCF7 from the baseline (Fig. 4F).In conclusion, we present strong evidence to support that, in the RAB4A signaling chain of stemness regulation, NUMB is downstream of RAB4A but upstream of NOTCH1, RAC1, and SOX2.Phenotypically, in both RAB4A high/NUMB low and RAB4A low/NUMB high cancer cells, NUMB suppresses the long-term sphere formation-a hallmark of cancer stemness. NOTCH1 is upstream of RAC1 and SOX2 in the RAB4A-NUMB signaling pathway that promotes cancer stemness The data presented thus far demonstrates that NUMB is essential for RAB4A regulation of stemness, and that it does so through NOTCH1, RAC1, and SOX2.Here we further assessed the position of NOTCH1 in the signaling chain of RAB4A regulation.To this end, we performed a rescue experiment by expressing NICD-the transcription activator fragment of NOTCH1-in RAB4A knockdown MDA-MB-231 cells.When exogenous NICD was introduced in the RAB4A knockdown cells, the SOX2 level recovered to the comparable level as control cells (Fig. 5A).From the qPCR analysis, we observed that NICD expression restored the transcript level of SOX2, consistent with the role of NICD in the transcription Fig. 4 RAB4A regulates cancer stemness through suppression of NUMB, which in turn modulates NOTCH1 and SOX2 expression and RAC1 activation.A Immunoblot analysis of NUMB, NOTCH1, and SOX2 protein levels in response to RAB4A knockdown alone and combined RAB4A and NUMB knockdown in MDA-MB-231 cells.Cell lysates and nuclear/cytosol fractionation were prepared for western blot from MDA-MB-231 cells expressing control shRNA (1), or RAB4A shRNA#1 (2), or concurrently RAB4A shRNA#1 and NUMB shRNA#1 (3), or concurrently RAB4A shRNA#1 and NUMB shRNA#2 (4), or RAB4A shRNA#2 (5), or concurrently RAB4A shRNA#2 and NUMB shRNA#1 (6), or lastly RAB4A shRNA#2 and NUMB shRNA#2 (7).The numeric label below each lane indicates the identity of the cell lysate as described above and will be used in panel (B) pulldown assay.B Pulldown of GTP-bound RAC1 to study the activation status of RAC1 in samples 1, 2, 3, 4, 5, 6, and 7 as described in (A).For each condition, the first lane is from the lysate incubated with GTPγS, the second lane is the lysate incubated with GDP, and the third lane is the sample without incubation with nucleotide to assess the quantity of the GTP bound form of RAC1 in the lysate.At the bottom of the panel is a blot of the lysate input.C Serial replating sphere formation assay on MDA-MB-231 cells with RAB4A knockdown alone and in combination with NUMB knockdown.The left side of the panel shows the representative microscopic images of the third-generation (Gen-3) plated spheres; the right side of the panel presents the quantification of spheres.D Immunoblot analysis of NUMB, NOTCH1, and SOX2 protein levels in response to NUMB knockdown in MCF7 cells.Cell lysates and nuclear/cytosol fractionation were prepared for western blot from cells expressing control shRNA (1), or NUMB shRNA#1 (2), and NUMB shRNA#2 (3).The numeric label below each lane indicates the identity of the cell lysate as described and will be used in (E).E Pulldown of GTP-bound RAC1 to study the activation status of RAC1 in samples 1, 2, and 3 as described in Panel (D).For each condition, the first lane is from the lysate incubated with GTPγS, the second lane is the lysate incubated with GDP, and the third lane is the sample without incubation with nucleotide.At the bottom of the panel is a blot of the lysate input.F Serial replating sphere formation assay on MCF7 cells with NUMB knockdown.The top of the panel shows the representative microscopic images of the third-generation (Gen-3) plated spheres; the bottom of the panel presents the quantification of spheres.Data from sphere formation assays (C, F) are presented as mean ± SEM (n ≥ 3); * Indicates when p < 0.05 compared to control.regulation (Fig. 5B) [46,47].We also noted a slight increase in the full-length NOTCH1 protein, supporting the notion that there exists some level of self-regulation of NOTCH1.Interestingly, we observe that NICD expression also affected NUMB at a modest level, suggesting a mutual regulatory mechanism between NUMB and NOTCH1 (Fig. 5A,B), albeit the NUMB control of NOTCH expression appears to be the dominant one (Fig. 4A,D).We also introduced NICD in the NUMB-high, NOTCH1-low MCF7 cells; here the expression of NICD elicited a strong SOX2 expression and a modest suppression of NUMB (Fig. 5C,D), consistent with the observations made in MDA-MB-231 cells.To place the order of NICD and RAC1 in RAB4A signaling, we evaluated RAC1 activity level in relation to NICD level and found that increasing NICD enhanced RAC1 activity (Fig. 5E,F), suggesting that NICD is a strong positive regulator of RAC1 activation. We then evaluated the phenotypic impact of NICD in RAB4A regulation of cancer cell stemness.As expected, knockdown RAB4A in high sphere-forming MDA-MB-231 cells abolished the third passage sphere formation, which was rescued by the expression of NICD to similar levels as the parental cells (Fig. 5G).For the low sphere-forming MCF7 cells, NICD expression dramatically increased the third-generation sphere formation (Fig. 5H).In summary, these studies conclude that NOTCH1, through its activation product NICD, forms a necessary link in the RAB4A signaling axis upstream of RAC1 and SOX2 in promoting the cancer cell stemness/self-renewal property.NUMB and NICD also appear to form a mutual regulation loop to exert a balance in controlling stemness. SOX2 is downstream of NUMB, NOTCH1, and RAC1 in mediating the signal from RAB4A in the regulation of cancer cell stemness/self-renewal So far, we have demonstrated that NUMB, NOTCH1, and RAC1 regulate cancer cell self-renewal downstream of RAB4A, and that this signaling chain controls the transcription of SOX2.However, the question of whether SOX2 is necessary and sufficient in the RAB4A regulation of cancer stemness still needs to be addressed.To this end, we performed a SOX2 rescue study in MDA-MB-231 RAB4A knockdown cells, and in RAB4A-low MCF7 parental cells, to determine whether the self-renewal could be boosted.We found that, introducing physiological levels of SOX2 expression exerted no impact on the levels of NUMB and NOTCH1 (Fig. 6A).The RAC1 activity assay demonstrated that SOX2 expression did not change the activation level of RAC1 in either MDA-MB-231 cells (Fig. 6B) or in MCF7 cells (Fig. 6C).From the sphere formation study, we found that SOX2 expression significantly rescued the sphere forming ability that was lost upon RAB4A knockdown in MDA-MB-231 cells (Fig. 6D).In MCF7 cells, SOX2 expression dramatically elevated sphere formation from the low level seen in parental cells (Fig. 6E).It was noted however that, distinct from the NUMB knockdown and NICD expression rescue in MDA-MB-231 cells that almost completely restored the sphere formation ability to the level before RAB4A knockdown (Figs.4C and5G), SOX2 expression partially rescued the sphere formation despite the restoration of SOX2 level to that similar to the parental cells (Fig. 6D).These observations suggest that downstream of RAB4A-RAC1 axis of signaling there may be additional mediator(s) besides/parallel to SOX2 to regulate self-renewal.Future studies should shed light on these additional signaling events. ", "section_name": "Suppressing NUMB expression reverses the effects of RAB4A knockdown on NOTCH1 and SOX2 expression, RAC1 activity, and the stemness phenotype", "section_num": null }, { "section_content": "The in vitro study of the signaling chain whereby RAB4A impacts cancer cell stemness has demonstrated that NUMB, NOTCH1, RAC1, and SOX2 are essential in mediating the regulatory effects of RAB4A on CSCs in multiple cancer cell lines of diverse tissue origins.Among these effects, we have already validated in the in vivo setting that constitutively active RAC1 is able to restore the lost tumor formation ability resulting from RAB4A knockdown [29]. Here, we sought to validate, in a xenograft mouse model, the essential role of NUMB, NOTCH1, and SOX2 in RAB4A-driven tumor formation.From parental MDA-MB-231 cells, we generated stable cell lines that contain the vector of (i) control, (ii) RAB4A shRNA, (iii) RAB4A shRNA and NUMB shRNA, (iv) RAB4A shRNA and NICD cDNA, and (v) RAB4A shRNA and SOX2 cDNA.The identities of these cells were confirmed by gene expression changes (Supplementary Fig. S6).These cells were deposited subcutaneously into the flanks of NOD SCID mice to observe tumor growth.All the mice were sacrificed when the tumors reached around 1 cm 3 according to the approved IACUC protocol; the representative images for each group are presented in Fig. 7A.Consistent with previous in vivo observation, effective RAB4A knockdown with either of the two shRNAs abolished the tumor-forming ability of MDA-MB-231 cells (Fig. 7A,B).Concurrent knockdown of NUMB largely restored the tumor formation, albeit with a 7-to 10-day delay compared to control cells (Fig. 7B).Expression of NICD, the nuclear fragment of NOTCH1 that is shown to be downstream of NUMB but also feeds back to NUMB, completely reversed the effect of RAB4A knockdown on the tumor forming ability (Fig. 7B).Similar to the observation made in the in vitro sphere formation assay, the expression of SOX2 partially but significantly restored the tumor forming ability, with about a 50-day delay in tumor growth (Fig. 7B).These in vivo rescue experiments confirm the signaling axis of RAB4A-NUMB-NOTCH-RAC1-SOX2 in the control of stemness/self-renewal that is the foundation for tumor formation (Fig. 7C).It is important to point out the discrepancy between the extent of rescue by SOX2 and NICD expression and NUMB knockdown, as well as CA-RAC1 expression [29], which suggests that there may be other regulator(s) acting parallel of SOX2 downstream of NUMB-NOTCH1-RAC1.Further investigation will likely complete this regulatory map. ", "section_name": "RAB4A-NUMB-NOTCH1-RAC1-SOX2 signaling is essential for tumor formation in vivo", "section_num": null }, { "section_content": "RAB4A is a master regulator of tumorigenesis and cancer progression RAB4A, a member of the RAB family of small GTPases, is wellrecognized for its essential function in regulating intracellular vesicle trafficking, particularly the short-loop endosome recycling, through which it plays important roles in cellular function [48].Relatively, RAB4A is less known for its role in cancer cell signaling; however, several functions have recently emerged in cancer invasiveness and growth.As a regulator of vesicular trafficking, RAB4A may contribute to tumor growth by modulating the transport of various signaling molecules such as growth factors and receptors to the cell surface, thereby affecting cancer cell proliferation and tumor progression [49,50].Our recent studies suggest that RAB4A influences the rearrangement of the cytoskeleton, affects the trafficking of integrins and cell adhesion properties, and regulates the EMT process which is considered the common foundation for cancer stemness and metastatic potential [29,42].Interestingly, a genetic mouse model demonstrated that the knockout of constitutively active RAB4A resulted in a depletion of immune cells and impaired receptor recycling [51].Hence, we speculate that RAB4A likely has immune regulatory function and may impact on tumor microenvironment, a major focus in cancer therapy development for many solid tumors.Despite these recent findings, there remains a major gap in knowledge of the molecular mechanisms underlying the roles of RAB4A in cancer.The work presented in this manuscript describes a step-by-step identification of a novel signaling axis RAB4A-NUMB-NOTCH1-RAC1-SOX2 to elucidate the transmission of signaling downstream from RAB4A to control stemness and tumor formation.Given its impact on cancer stemness and EMT, we postulate that suppressing RAB4A expression or inhibiting its function would likely halt tumor growth and metastasis, and hence is worthy of future evaluation as a therapeutic target.In this current study, we provide direct evidence, using both RAB4A-high and RAB4A-low cancer cell lines of diverse tissue origin, to demonstrate that suppressing RAB4A reduces the oncogenic potential in RAB4A-high cells, and that increasing the level of RAB4A promotes oncogenic potential in RAB4A-low cells.Furthermore, we conclude that the signaling axis of RAB4A-NUMB-NOTCH1-RAC1-SOX2-a previously unknown pathway-is the molecular mechanism important for tumorigenesis. Transcription regulation is the predominant mechanism of the RAB4A-to-SOX2 chain of signal transmission The RAB4A small GTPase is best known for its role in endocytic vesicle recycling, especially that of the so-called short-loop-recycling, transporting endocytosed cargos quickly back to the plasma membrane.In our recent study, we observed that integrin β3 recycling, and in turn its intracellular signaling, is subjected to RAB4A regulation [42].Hence, it is interesting that we found in this study that RAB4A plays a determinant role in NUMB transcription through which it controls NUMB protein level (Fig. 2).To evaluate whether the RAB4A regulation of NUMB expression is at the transcription or at the post-transcriptional processing level, we performed the classical Actinomycin D treatment study.Consistent with a role on transcription, we found that in the presence of Actinomycin D, the baseline NUMB transcript level is reduced but the half-life of the transcript remains the same with and without RAB4A knockdown (Supplementary Fig. S7A).Notably, this effect is ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "Gen ", "section_name": "MDA-MB-231 MCF7", "section_num": null }, { "section_content": "Fig. 6 SOX2 expression restores the sphere-forming ability that was lost from RAB4A knockdown.A Immunoblot analysis of NUMB, NOTCH1, and SOX2 protein levels in response to SOX2 expression in MDA-MB-231 and MCF7 cells.For MDA-MB-231 cells, the lysates and nuclear/cytosol fractionation were prepared for western blot from cells expressing control shRNA (1), or RAB4A shRNA#1 (2), or concurrently RAB4A shRNA#1 and SOX2 cDNA (3), or RAB4A shRNA#2 (4), or concurrently RAB4A shRNA#2 and SOX2 cDNA (5).The label below each lane indicates the identity of the cell lysate as described above.For MCF7 cells, the lysates and fractionation were prepared from cells with and without exogenous SOX2 expression.B Pulldown of GTP-bound RAC1 to study the activation status of RAC1 in samples 1, 2, 3, 4, and 5 as described in (A).For each condition, the first lane is from the lysate incubated with GTPγS, the second lane is the lysate incubated with GDP, and the third lane is the sample without incubation with nucleotide, therefore assessing the quantity of the GTP bound form of RAC1 in the lysate.At the bottom of the panel is a blot of the lysate without a pull-down to show the quantity of RAC1 in the input.C Pulldown of GTPbound RAC1 to study the activation status of RAC1 of the lysate from MCF7 cells with or without exogenous expression of SOX2.D Serial replating sphere formation assay on MDA-MB-231 cells with RAB4A knockdown alone and in combination with exogenous SOX2 expression.The left side of the panel shows the representative microscopic images of the third-generation (Gen-3) plated spheres; the right side of the panel presents the quantification of spheres.E Serial replating sphere formation assay on MCF7 cells with and without exogenous SOX2 expression.The top images are representative microscopic photos and the bottom shows the analysis from three biological repeats of the third generation sphere.Data analysis of (D, E) are from three biological repeats and presented as mean ± SEM (n ≥ 3); * Indicates when p < 0.05 compared to control. likely not directly downstream of RAB4A but through intermediates, therefore warranting further studies to define.We also found that NUMB regulates the expression level of NOTCH1 at the transcript level (Fig. 2D-H).Interestingly, prior to our findings, numerous reports described NUMB regulates NOTCH1 trafficking and degradation in the endosome; however, the role of NUMB in NOTCH1 transcription has not been described [52,53].Hence, we further investigated the role of NUMB in regulating NOTCH expression by both Actinomycin D treatment and luciferase reporter assays for the impact of both RAB4A and NUMB on NOTCH1 promoter transcription [44].We found that RAB4A knockdown led to a reduction of basal NOTCH transcript level that was reversed by concurrent knockdown of NUMB.However, neither RAB4A knockdown alone nor combined RAB4A and NUMB knockdown affected the half-life of the NOTCH1 transcript (Supplementary Fig. S7B).These data suggest transcription regulation of NOTCH1 by RAB4A through NUMB.Luciferase reporter assay using NOTCH1 promoter with control KD, RAB4A KD alone, and concurrent RAB4A and NUMB KD provides further support for this notion (Supplementary Fig. S7C,D).To look further into the contribution of endosome-related mechanisms in the regulation of NUMB and NOTCH, we evaluated the impact of knocking down RAB5A on the levels of NUMB, NOTCH/NICD, and SOX2.Since RAB5A is the \"yin\" to the RAB4A \"yang\" in endocytosis, we would expect a change in the level of these proteins if endocytosis plays a significant role in their levels.Here, though, we did not observe any effect of RAB5A on the levels of NUMB, NOTCH, and SOX2 (Supplementary Fig. S8).These studies support the conclusion that the RAB4A-NUMB regulation of NOTCH1 is mainly through transcriptional control.We anticipate that future investigation will provide further details on the nature of NUMB control of transcription of NOTCH1.We acknowledge that the RAB4A-NUMB-NOTCH1-RAC1 -SOX2 signaling axis as detailed in this work is not linear at every step.There clearly exists a mutual regulation between NUMB and NOTCH1 in that, although the predominant regulation is transmitted from NUMB to NOTCH1, the expression of NOTCH1 at the physiological level also exerts a negative feedback on NUMB.Notably, the mutual regulation is both at the transcriptional level, which will be an interesting subject in future studies. SOX2 is likely not the only effector downstream of RAB4A-NUMB-NOTCH1-RAC1 signaling in the regulation of stemness Finally, our data point to the existence of other effector(s) downstream of RAC1 besides SOX2 in the control of stemness.This assessment is based on the rescue studies that position the up-and downstream effectors in this pathway, in which we observed that NUMB knockdown and NICD expression nearly completely reversed the loss-of-function phenotypes of RAB4A knockdown both in vitro and in vivo and restored RAC1 activation to the original level.In this regard, in our recent study, we also found that expressing activated RAC1 completely restored the sphere formation and tumor formation ability lost due to loss-of-RAB4A [29].In contrast, restoring SOX2 expression back to the baseline level of that before RAB4A knockdown only partially rescued the serial-replating sphere formation and tumor formation, which suggests that downstream of RAC1 the regulation of stemness is likely transmitted through multiple effectors, with SOX2 being a predominant one.It will be interesting to identify in future studies other RAC1 downstream effectors that transmit signals for cancer stemness regulation. Overall, this study identified a novel regulatory axis, RAB4A-NUMB-NOTCH1-RAC1 in regulating cancer stemness and tumor initiation and progression and these targets may be further investigated for drug development and cancer therapeutics. ", "section_name": "MDA-MB-231 MCF7 GTPgS + --+ --+ --GDP -+ --+ --+rec-RAC1 ---------+ GTPgS + --+ --GDP -+ --+rec-RAC1 ------+", "section_num": null } ]	[ { "section_content": "The study is supported by the National Medical Research Council's Individual Research Grant CIRG/1486/2018 and MOH-000944; the Ministry of Education of Singapore Tier2 fund MOE2017-T2-1-039 and Ministry of Education of Singapore Tier2 fund MOE2018-T2-1-147; and the Khoo Postdoctoral Fellowship Award, Duke-NUS-KFPA/2020/0041, Singapore. ", "section_name": "FUNDING", "section_num": null }, { "section_content": "The data supporting the findings of this study are available from the corresponding author upon reasonable request. ", "section_name": "DATA AVAILABILITY", "section_num": null }, { "section_content": "SK and MW designed the studies, collected and/or assembled, analyzed, and interpreted the data.PJC analyzed and interpreted data.MW conceived the project, applied and acquired funding, and provided administrative support.All authors wrote and approved the manuscript. The authors declare no competing interests. All methods performed in the studies were in accordance with the relevant institutional guidelines and regulations.The ethics approval for animal work was provided by the Institutional Animal Care and Use Committee (IACUC) of Duke-NUS Medical School (protocol no.2021/SHS/1627).There were no human tissues or human subjects employed for the studies. The online version contains supplementary material available at https://doi.org/10.1038/s41419-024-07172-w. Correspondence and requests for materials should be addressed to Mei Wang. Reprints and permission information is available at http://www.nature.com/reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "SK and MW designed the studies, collected and/or assembled, analyzed, and interpreted the data.PJC analyzed and interpreted data.MW conceived the project, applied and acquired funding, and provided administrative support.All authors wrote and approved the manuscript. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "The authors declare no competing interests. ", "section_name": "COMPETING INTERESTS", "section_num": null }, { "section_content": "All methods performed in the studies were in accordance with the relevant institutional guidelines and regulations.The ethics approval for animal work was provided by the Institutional Animal Care and Use Committee (IACUC) of Duke-NUS Medical School (protocol no.2021/SHS/1627).There were no human tissues or human subjects employed for the studies. ", "section_name": "ETHICS APPROVAL AND CONSENT TO PARTICIPATE", "section_num": null }, { "section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41419-024-07172-w. Correspondence and requests for materials should be addressed to Mei Wang. Reprints and permission information is available at http://www.nature.com/reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "ADDITIONAL INFORMATION Supplementary information", "section_num": null } ]
10.1186/1747-1028-2-9	FBXW7/hCDC4 controls glioma cell proliferation in vitro and is a prognostic marker for survival in glioblastoma patients	<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>In the quest for novel molecular mediators of glioma progression, we studied the regulation of <jats:italic>FBXW</jats:italic> 7 (h<jats:italic>CDC</jats:italic> 4/h<jats:italic>AGO</jats:italic>/<jats:italic>SEL</jats:italic> 10), its association with survival of patients with glioblastoma and its potential role as a tumor suppressor gene in glioma cells. The F-box protein Fbxw7 is a component of SCF<jats:sup>Fbxw7</jats:sup>, a Skp1-Cul1-F-box E3 ubiquitin ligase complex that tags specific proteins for proteasome degradation. <jats:italic>FBXW</jats:italic> 7 is mutated in several human cancers and functions as a haploinsufficient tumor suppressor in mice. Any of the identified targets, Cyclin E, c-Myc, c-Jun, Notch1/4 and Aurora-A may have oncogenic properties when accumulated in tumors with <jats:italic>FBXW</jats:italic> 7 loss.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>We tested the expression of <jats:italic>FBXW</jats:italic> 7 in human glioma biopsies by quantitative PCR and compared the transcript levels of grade IV glioma (glioblastoma, G-IV) with those of grade II tumors (G-II). In more than 80% G-IV, expression of <jats:italic>FBXW</jats:italic> 7 was significantly reduced. In addition, levels of <jats:italic>FBXW</jats:italic> 7 were correlated with survival indicating a possible implication in tumor aggressiveness. Locus 4q31.3 which carries <jats:italic>FBXW</jats:italic> 7 was investigated by <jats:italic>in situ</jats:italic> hybridization on biopsy touchprints. This excluded allelic loss as the principal cause for low expression of <jats:italic>FBXW</jats:italic> 7 in G-IV tumors. Two targets of Fbxw7, Aurora-A and Notch4 were preferentially immunodetected in G-IV biopsies. Next, we investigated the effects of <jats:italic>FBXW</jats:italic> 7 misregulation in glioma cells. U87 cells overexpressing nuclear isoforms of Fbxw7 lose the expression of the proliferation markers PCNA and Ki-67, and get counterselected <jats:italic>in vitro</jats:italic>. This observation fits well with the hypothesis that Fbxw7 functions as a tumor suppressor in astroglial cells. Finally, <jats:italic>FBXW</jats:italic> 7 knockdown in U87 cells leads to defects in mitosis that may promote aneuploidy in progressing glioma.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Our results show that <jats:italic>FBXW</jats:italic> 7 expression is a prognostic marker for patients with glioblastoma. We suggest that loss of <jats:italic>FBXW</jats:italic> 7 plays an important role in glioma malignancy by allowing the accumulation of multiple oncoproteins and that interfering with Fbxw7 or its downstream targets would constitute a new therapeutic advance.</jats:p> </jats:sec>	[ { "section_content": "Glioblastoma (glioma grade IV, G-IV), the most common tumor arising in the central nervous system, is one of the deadliest cancers with a mean survival of less than one year [1].Reliable molecular predictors of survival outcome as well as novel targets for efficient therapy are urgently needed to improve life conditions of patients with glioma. In this study, we investigated the expression of FBXW7 (also known as hCDC4, hAGO, SEL10) in glioma.FBXW7 encodes one of the 75 F-box proteins identified so far in mammals [2].F-box proteins represent the variable receptor component of Skp1-Cul1-F-box (SCF) complexes, that mediates binding and ubiquitination of specific proteins, which are consequently recognized and destroyed by the proteasome.In contrast to other SCF complexes such as SCF Fbxl1 which target both positive and negative regulators of the cell cycle [3], all known targets of SCF Fbxw7 -namely Cyclin E [4][5][6], c-Myc [7], c-Jun [8], Notch 1 and 4 [9][10][11] and Aurora-A [12] -are cell growth promoters and potential oncoproteins.Their turn-over can thus be seen as an ultimate process in tumor suppression control.Indeed, FBXW7 itself behaves as a haploinsufficient tumor suppressor gene: the loss of one functional allele is enough to promote epithelial tumor growth in a mouse model [13].Given the number of its targets and the fact that FBXW7 is translated into three different isoforms with distinct subcellular localization, possible mechanisms of tumor suppression are bound to be complex and variable depending upon the cell type in which downregulation occurs. FBXW7 is mutated in many cancer cell lines and human tumors such as endometrial, pancreatic and colorectal cancers [6,[14][15][16][17].Mutations of FBXW7 in brain tumors have not been investigated yet, however the corresponding locus -4q31.3-belongs to the most frequently lost portion of chromosome 4 in glioblastoma [18,19].As far as targets are concerned, much attention has been focused on the misregulation of Cyclin E [20] and c-Myc [21] whereas other substrates such as Aurora-A and Notch receptors have not yet been comprehensively investigated.In tumors, correlations have been demonstrated between loss of function of FBXW7 and high levels of Cyclin E [20], and between Cyclin E overexpression and chromosomal instability [17].However, the overall effect of FBXW7 loss in cancer cells is not straightforward and seems to vary according to cancer types [16,22].Fujii and coworkers recently reported the accumulation of multiple targets in several FBXW7 mutant cell lines with a variable extent of increase in expression and even identified a different pattern of accumulation for two ovarian cancer lines, one accumulating predominantly both Cyclin E and c-Myc, the other Aurora-A solely [23]. In this study, we investigated the possible misregulation of Aurora-A and Notch 4 expression in glioma.Aurora-A is a mitotic kinase required for G2-M transition, centrosome maturation and alignment of chromosomes at metaphase.Its degradation by the proteasome occurs promptly after metaphase-anaphase transition and is necessary for mitotic exit [24].In normal cells, the expression of Aurora-A is thus rhythmic, discrete and detected in dividing cells only.By contrast, in cancer cells, Aurora-A is frequently overexpressed indicating its possible involvement in tumorigenesis [25].Indeed, Aurora-A overexpression results in centrosome amplification and cytokinesis failure -both promoting tetraploidization -and transformation of cells with already acquired checkpoint defects [26,27].How does Aurora-A accumulate in cancer cells?In bladder cancer, AURKA/STK15, the gene encoding Aurora-A at locus 20q13.2 is commonly amplified and the resulting overexpression is correlated with critical clinical parameters such as invasion, metastasis and poor survival [28].But the overexpression of Aurora-A is also frequently seen in tumors with no gain of AURKA at the DNA level suggesting that other deregulated mechanisms such as transcriptional activation or failure of a proteolysis component are responsible for Aurora-A accumulation.This may well apply to glioma: Klein and coworkers detected amplification of AURKA in 26% of malignant glioma while as much as 67% of the samples eventually overexpressed the gene [29]. Unlike all other targets of Fbxw7, Notch4 has not yet been investigated in human cancers though the protein is oncogenic when induced in the MMTV (mouse mammary tumor virus) mouse model [30].Thus far, Notch4 has been described as a vascular endothelium specific signaling receptor.Together with its ligand Delta-like 4 (Dll4), it is involved in vessel development by mediating arterial/ venous specification and vascular remodeling during embryogenesis [31].In tumors, Notch4 signaling is activated in the endothelium and is here again responsible for vascular maturation [32].On the other hand, it has been recently shown that NOTCH4 transcription can be derepressed in non-endothelial cells: in HeLa cells treated with endothelial growth factors, cell-type-specific AP-1 (activator protein 1) complexes are activated that are able to reprogram NOTCH4 expression [33].This raises the possibility that tumor cells, which produce high amounts of proangiogenic factors -like malignant glioma cells -may ectopically express NOTCH4. Here we report that expression of FBXW7 is strongly reduced in G-IV tumors, mostly independent of locus depletion, and that the levels of FBXW7 expression correlate with patient survival.We find that Aurora-A and Notch4 accumulate in perivascular zones of patient glioblastoma.Proliferation is significantly impaired in glioma cells overexpressing nuclear FBXW7 in vitro suggesting that it acts as a tumor suppressor in astroglial cells.Finally we show that knocking down FBXW7 in cultured glioma cells destabilizes chromosome segregation at mitosis, a process controlled by several targets of SCF Fbxw7 including Aurora-A. ", "section_name": "Background", "section_num": null }, { "section_content": "Expression of FBXW7 in glioma patients and correlation with survival FBXW7 expression was evaluated by quantitative RT-PCR in 56 G-IV and 5 G-II gliomas.G-IV tumors were classified according to the normalized expression levels of FBXW7 compared to the mean of expression levels in G-II tumors.FBXW7 is significantly downregulated (<0.75) in more than 80% G-IV tumors.As illustrated in Fig. 1A, it is severely decreased of more than 2-fold (<0.5) in most samples (73,2%).Correlation was next established between survival time after initial diagnosis of glioblastoma and FBXW7 expression levels.As shown in Fig. 1B, expression levels were heterogeneous, but when classified into two groups (<0.5 and ≥0.5), a significant difference of survival time was highlighted (Mann-Whitney U-test, P = 0.0099).Interestingly, out of 4 tumors collected from long survivors with G-IV (> 1000 days), 3 displayed normal FBXW7 levels.Patients classified in the two groups as in Fig. 1B were next subjected to Kaplan-Meier survival analysis illustrating that the outcome for patients with >0.5 levels of FBXW7 is significantly better than for patients with reduced expression (Fig. 1C).The same study performed with two related F-box protein encoding genes, FBXO6 and FBXL1/SKP2, showed a similar heterogeneous spectrum of expression but no evidence for significant correlation with survival (not shown).While this study was being conducted, Bredel et al. described an informative gene profiling of gliomagenesis, from which FBXW7 emerges as a downregulated inhibitor of the MYC interacting pathway [34], reinforcing our findings that it is a highly significant predictor of aggressiveness.Our results further demonstrate that FBXW7 can be used as a specific prognostic marker of survival for patients with glioblastoma.Together with other recently identified molecular predictors, FBXW7 may enhance comprehensive classification and molecular characterization of gliomas with a likely impact on clinical management including prediction of therapeutic response. ", "section_name": "Results and Discussion", "section_num": null }, { "section_content": "In p53+/-mice, the loss of one allele of FBXW7 get preferentially selected in radiation-induced lymphoma cells and promotes tumor development so that FBXW7 meets the criteria of a haploinsufficient tumor suppressor gene [13]. In the same study, the authors show that FBXW7 is a direct transcriptional target of p53 itself.This raises the possibil-ity that monoallelic deletion on its own may account for reduced expression of FBXW7 in patient tumors with active p53 (around 30% of primary glioblastoma [35]).FBXW7 maps on 4q31.3.Analyses of this region have been recently conducted in esophageal adenocarcinoma and identified deletions in 40% of tumors.The sequence of the remaining allele was wild-type in most of the cases [36].Further studies should confirm the functional implication of monoallelic deletion (rather than loss of heterozygosity) -i.e. the haploinsufficiency of FBXW7 -in this type of cancer.We sought for 4q31.3 deletions in our cohort of G-IV patients.The locus specific probe containing the complete FBXW7 gene sequence was selected from the RP11 BAC library (clone 300I24) and proved to hybridize specifically at 4q31 on metaphase normal chromosomes (Fig. 2A).Twenty-eight tumors were subjected to touchprint dual color FISH analysis using the FBXW7 locus specific probe and a chromosome 4 subcentromeric probe for chromosome copy number assessment.The patterns of signal distribution in individual nuclei were collected for 50-100 cells per tumor.Representative images are shown in Fig. 2B-D.Results of this analysis are illustrated in the table of Fig. 2. Samples 1 and 2 were tumors with no significant downregulation of FBXW7 and did show no deletion of 4q31.3.FISH analysis identified FBXW7 hemizygous deletions in 31% (8/26) cases with downregulation.In these cases however, only a minority (10-23%) of cells exhibited a 4q31.3loss while other nuclei were mainly disomic for chromosome 4 with 2 copies of 4q31.3 (see individual patterns in table of Fig. 2).FISH analysis did not detect any imbalanced case so that 69% of the tumors (18/26) were classified as nondeleted.This analysis suggests that only for a minority of glioblastoma, monoallelic deletion of 4q31.3 may participate in reduction of FBXW7 expression.If downregulation is required for glioblastoma progression, these events may be selected preferentially in tumor cells that retain P53 expression.Nonetheless, we conclude that allelic loss is not a recurring cause for reduced FBXW7 expression in glioblastoma. ", "section_name": "Allelic loss is not the primary cause for low expression of FBXW7 in G-IV tumors", "section_num": null }, { "section_content": "We next asked if the loss of function of Fbxw7 protein may participate in glioma malignancy by causing accumulation of specific targets.Protein extracts from the biopsies were examined by western blotting using anti-CyclinE, anti-c-Myc, anti-Aurora-A and anti-Notch4 antibodies.Generally, levels of detected proteins were variable between samples and no significant correlation with FBXW7 expression could be made.It was striking however, that Aurora-A was easily detected in over 81% (35/ 43) of the samples with significant decrease in FBXW7 expression, whereas it failed under detection threshold in Expression of FBXW7 in glioma and correlation to survival Figure 1 Expression of FBXW7 in glioma and correlation to survival.(A) FBXW7 expression was measured by quantitative RT-PCR in 56 G-IV compared to a pool of 5 G-II.73% of high grade tumors show very significant low levels of FBXW7 transcripts (<0.5).(B) Relative levels of FBXW7 were heterogeneous, but expression defines two groups of patients with different survival times after diagnosis.The median survival in the group with FBXW7 levels >0.5 was 490 days, whereas patients with low expression (<0.5) had a median survival of 335 days.The difference between the two groups is significant (P = 0.0099, Mann-Whitney U-test, two-tailed).(C) Patients classified in the two groups as shown in B were subjected to Kaplan-Meier survival analysis.Survival curves for the two patient groups differed significantly (P = 0.011; Log-rank test).Note that two patients in the >0.5 group were still alive at the time the analysis were performed, more than 2000 days after initial diagnosis. G-II with consistent FBXW7 signals (Fig. 3A).The same tendency was observed for Notch4 but neither for Cyclin E, nor c-Myc (not shown).This prompted us to investigate the presence of Aurora-A and Notch4 on sections of gliomas of different histological grades.In G-II, Aurora-A was detected sparsely in less than 5% of the tumor cells (Fig. 3B) as expected from the low proliferation index (Fig. 3E) and accumulated only in a few tumor areas (Fig. 3C).Notch4 was strongly associated with α-SMA (alpha smooth muscle cell actin) positive vessels (Fig. 3D), as expected from its previously reported role in vessel maturation [37], but was not detected in any proliferating tumor cells (Fig. 3E).In contrast, both Aurora-A and Notch4 were strongly detected in distinct areas of aggressive primary glioblastoma (Fig. 3, lower panel).Aurora-A massively accumulates in tumor cells surrounding vessels (Fig. 3G &3J).Anti-Notch4 antibody also stains tumor cells preferentially around vessels in glioblastoma in addition to the endothelial lining of α-SMA positive arterioles (Fig. 3H &3K).No accumulation of any of the two proteins was seen in poorly vascularized tumor areas (not shown). All in all, two oncoproteins the mitotic kinase Aurora-A and the signaling receptor Notch4, which levels depend on FBXW7, are strongly expressed in specific zones of G-IV tumors, particularly around blood vessels. .By contrast, in invasive primary G-IV (lower panel) of known histology and α-SMA labeling (F, I) discriminating vascular structures (v, vein ; a, arteriole), Aurora-A is massively detected in specific zones of G-IV, mostly around vessels (G-J).Notch4 is also detected in some tumor cells in addition to the endothelial lining of arterioles (H-K). ", "section_name": "Aurora-A and Notch4 accumulate in G-IV tumors", "section_num": null }, { "section_content": "The three isoforms of Fbxw7, α, β, and γ, have a conserved structure and molecular function but differ in their subcellular localization which is determined by the first alternative exon.Welcker and coworkers reported that, in U2OS cells, α-Fbxw7 is strongly detected in the nucleus, β-Fbxw7 in the cytoplasm and γ-Fbxw7 is mostly associated with nucleoli [21].Hence, the three isoforms are likely to mediate degradation of specific targets in a localizationdependent manner.In order to study the effect of FBXW7 misregulation in glioma, U87 cells were transiently transfected with plasmids encoding each FLAG-tagged Fbxw7 isoform.Transfected cells were detected by immunofluorescence using anti-FLAG antibody.As shown in Fig. 4A-C, the subcellular localization of α-Fbxw7 and β-Fbxw7 was similar to that previously reported for other cell lines (ie, nucleoplasmic and cytoplasmic respectively) whereas isoform γ localization varied according to its level of expression: weak expression was restricted to the nucleoli whereas stronger expression was found in the cytoplasm and/or in the nucleus.We next asked whether Fbxw7 had an effect on glioma cell proliferation.Cells that express the proliferation markers PCNA or Ki-67 were scored among FLAG-positive cells compared to FLAG-negative cells.As illustrated in Fig. 4D-J, immunodetection of PCNA was specifically reduced in cells overexpressing nuclear Fbxw7.Cells overexpressing nuclear isoforms α and γ also display strongly reduced expression of Ki-67 (P < 0.0005 and P < 0.01 respectively) whereas expression of Ki-67 did not significantly change in cells overexpressing the cytoplasmic isoform β (Fig. 4K). The effect of Fbxw7 on proliferation was further confirmed by showing that nuclear Fbxw7 counterselects transiently transfected cells in a time-course culture (Fig. 4L): the growth of cells overexpressing α-Fbxw7 or γ-Fbxw7 is strongly inhibited between 24 and 72 h after transfection (P < 0.0001 and P < 0.005 respectively) whereas FLAG negative internal control cells expand of more than 2-fold.By contrast, overexpression of isoform β does not significantly affect the growth of U87 cells.FLAG positive cells were screened for Aurora-A expression by double immunofluorescence with anti-FLAG and anti-Aurora-A antibodies.Amongst cells expressing FBXW7 in the nucleus (n > 400) no Aurora-A staining was detected compared to untransfected cells out of which 10% were Aurora-A positive.This mutually exclusive expression pattern suggests that Fbxw7 targets Aurora-A in glioma cells.This may interfere with cell cycle checkpoint hence the dramatic reduction of dividing cells.Given the effect of Aurora-A knockdown, we can anticipate that cells may then be arrested in the next cycle by the post-mitotic checkpoint [25], hence their progressive counterselection in culture. In summary, these results indicate that nuclear Fbxw7 exerts an inhibitory effect on proliferation of glioma cells in vitro and therefore support the hypothesis that loss of nuclear FBXW7 expression might contribute to tumor progression in patients with astroglial tumors. ", "section_name": "Overexpression of FBXW7 inhibits proliferation of U87 cells in vitro", "section_num": null }, { "section_content": "In order to examine the consequences of FBXW7 downregulation in glioma, U87 cells -that express low levels of FBXW7 in culture (not shown) -were transfected with previously described specific siRNAs directed against a shared exon of FBXW7 [20].Cells transfected with either control siRNA or FBXW7 siRNA were grown for 48 hours and stained with DAPI and anti-Aurora-A antibody.Nuclei with individualized chromosomes and Aurora-A positive staining [24] were classified as mitotic and accounted for at least 4% of cells in independent control experiments.We compared mitotic figures in FBXW7 knockdown cells to control cells (>100 mitosis for each group) and found that 98.5% of the figures were normal in controls and could be assigned to a specific phase as in Fig. 5A.By contrast, as much as 16% of mitosis in FBXW7 knockdown cells were seen with 3 or 4 spindle poles (Fig. 5B), and there was significantly less normal metaphases (Fig. 5C).Unexpectedly, the number of mitotic figures was significantly decreased in FBXW7 knockdown cells.This suggests that in glioma cells, loss of Fbxw7 function primarily triggers mitotic defects that could select for aneuploid cells with growth advantage in the evolving tumor. Possible targets that accumulation may cause checkpoint disturbances and chromosomal instability are Aurora-A and Cyclin E although they could not be quantitatively monitored in these transient transfection assays.Cyclin E accumulation has been shown to raise similar defects in colorectal cancer cells [17] and is accumulated in tumors of different types [20] including glioma with poor prognosis [38]. ", "section_name": "Fbxw7 knockdown causes mitotic defects in U87 cells", "section_num": null }, { "section_content": "In this study, we have shown that the expression of FBXW7 is strongly reduced in glioblastoma.Moreover, FBXW7 emerges as a relevant survival marker for patients with glioblastoma that warrants further validation in the clinic.Our results suggest that FBXW7 downregulation promotes gliomagenesis via the accumulation of oncogenic cell cycle regulators that control cell division such as Aurora-A and/or Cyclin E or Notch.The contribution of each of the targets remains to be investigated in details in glioma. Gene therapy with FBXW7 is already foreseen for cancer [23].Glioblastoma patients should benefit of an ameliorated survival prognosis based on FBXW7 quantification.Future studies will tell if there is also a way to improve Effect of FBXW7 overexpression in glioma cells Figure 4 Effect of FBXW7 overexpression in glioma cells.Immunodetection of FLAG-Fbxw7 (green) in U87, 48 hours after transfection with plasmids encoding each of the three isoforms α (A), β (B), γ (C).Nuclei are labeled with DAPI (blue) and actin cytoskeleton with rhodamin-coupled phalloidin (red), bar = 50 μm.α-Fbxw7 localizes to the nucleus, β-Fbxw7 is cytoplasmic, γ-Fbxw7 localization varies.It is restricted to the nucleolus at low levels (insert) and leaks in the nucleus and in the cytoplasm at higher doses.Example of PCNA detection (red) in U87 cells overexpressing FLAG-Fbxw7 (green) α (D), β (E), γ (F).In lower panels (G-I), only the red signal in the same fields is shown.An example of α-Fbxw7-positive PCNA-negative cell is shown with arrowhead.bar = 10 μm.Quantification analysis of PCNA expression 48 h after transfection (J).Scoring was established on >150 cells per assay.The results (mean of two independent experiments, ± SEM) are expressed as the ratio of PCNA + cells in FLAG + versus FLAG -cells from the same transfection well.Quantification analysis of Ki-67 expressing cells in FLAG -and FLAG + cells 48 h after transfection (K).Statistical analysis was performed using the Fisher's exact test (n > 150 cells for each group).The experiment was repeated twice with similar results.Cell growth after transfection with each isoform expression plasmid (L).For each assay, cells were stained with DAPI and anti-FLAG antibody 24 h and 72 h after transfection.FLAG positive and negative cells were scored from 20 independent 40×-magnified fields and their 72 H/24 h ratio compared.Cells overexpressing nuclear isoforms α and γ are significantly counterselected.Statistical analysis was performed using the Fisher's exact test (n > 1000 cells for each assay).The experiment was repeated twice and analyzed once after 48 h with similar results. therapy based on Fbxw7 rescue for those with the most dismal prognosis. ", "section_name": "Conclusion", "section_num": null }, { "section_content": "", "section_name": "Methods", "section_num": null }, { "section_content": "U87 human cells glioma (ATCC/LGCpromochem, Molsheim, France) were maintained in DMEM with 10% FBS, antibiotics, and L-glutamine.Glioma biopsies were classified according to the World Health Organization [39], the median survival of the G-IV group was 379 days (n = 54 patients, excluding 2 patients still alive at the time of analysis).Samples were immediately snap-frozen and stored until further use for protein and RNA purification, tissue sectioning, and touch printing of interphase nuclei.All procedures complied with current French laws. ", "section_name": "Cells and tissue collection", "section_num": null }, { "section_content": "The FBXW7 probe contained in BAC clone RP-11 300I24 was labeled by nick translation with SpectrumGreen dUTP (Abbott-Vysis, France) and co-hybridized with a chromosome 4 alpha-satellite probe (D4Z1) coupled to rhodamin (Qbiogene, Illkirch, France).Efficiency and specificity were assessed on metaphases and interphases from cultured human lymphocytes.Dual color FISH analysis were performed on touchprints of frozen tissues as previously described [40].The slides were then fixed by immersion in methanol and methanol/acetic acid baths.After drying and dehydratation, the probes were added to each slide.A coverslip was placed over each hybridization slide and sealed with rubber cement.Slides and probes More than 3400 cells were analyzed for each condition.Aurora-A positive cells as in A and B were classified as mitotic and accounted for around 4% of total cells in controls.FBXW7 siRNA-transfected cells showed significantly less mitotic figures (72.2% of controls, P = 0.0094), fewer normal metaphases (27.5% of controls, P = 0.022), but much more abnormal mitoses (7.3 fold compared to controls, P = 0.0042; unpaired t-test, two-tailed). ", "section_name": "Fluorescence in situ hybridization (FISH) on interphase tumor nuclei", "section_num": null }, { "section_content": "were co-denatured using the Hybrite Hybridization System (Abbott-Vysis, Rungis, France) at 73°C during 5 min.Hybridization was performed 24 h at 37°C in a humidified box.Finally, slides were washed, dehydrated and nuclei were counterstained with 4,6-diamidino-2phenyindole (DAPI) diluted in Vectashield (Abcys, Paris, France).The microscopic analysis was done by two independent observers using a fluorescent Axioplan II microscope (Zeiss, Le Pecq, France).A minimum of 50 tumour cell nuclei were evaluated for each slide.Hybridization signals of control (centromere of chromosome 4, CEN4) and test (FBXW7) probes were counted for each nucleus.Nuclei were then classified either as 1) deleted (ratio of control and test probes 2/1, 4/2, 3/1, 4/1,...), 2) imbalanced (disproportion of the ratio of control and test probes such as 3/2, 4/3, 5/3, etc...), or 3) non-deleted (equal ratio of control and test probes signals).The cut-off value was determined as the mean + three standard deviations of the percentage of deleted nuclei on control tissues [41].Finally, a tumor was classified either as deleted (% of deleted nuclei ≥ cut-off), or as non-deleted (% of deleted + imbalanced nuclei < cut-off) or as imbalanced (% of imbalanced nuclei or sum of imbalanced + deleted nuclei ≥ cut-off). ", "section_name": "Effect of FBXW7 knockdown in glioma cells", "section_num": null }, { "section_content": "RNA was purified from biopsies or cultured cells using RNeasy columns (Qiagen, Courtaboeuf Cedex, France).RNA quality was checked by electrophoresis and any sample displaying degraded RNA was excluded from the study.RNA was reversed-transcribed with SuperScript II RNase H-Reverse Transcriptase (Invitrogen, Cergy Pontoise Cedex, France) by using oligo (dT) 15 priming.Humanspecific primers for qPCR were designed and evaluated for amplification efficiency.Primer sequences were: α-tubulin (NM_006082), 5'-GAGTGCATCTCCATCCACGTT-3', 5'-TAGAGCTCCCAGCAGGCATT-3', FBXW7 (target sequence common to all isoforms), 5'-CCACTGGGCTTG-TACCATGTT-3', 5'-CAGATGTAATTCGGCGTCGTT-3'.Real-time PCR was carried out in a MX3000P thermocycler (Stratagene) by using SYBR Green dye (ABgene, Courtaboeuf Cedex; France).FBXW7 expression was steady in all G-II samples tested (mean ΔCt = 6.27,SD = 1).Hence, five controls were individually run in parallel with every G-IV PCR assay, and the mean ΔCt served as normalization value.Normalization and quantification were calculated as described previously [42].All G-IV samples were tested in a minimum of two independent experiments. Cell transfection U87 cells were seeded in LabTek chambers (100000 cells per well) and transfected the following day with 100 ng (α) or 500 ng (β and γ) of plasmid containing FLAG-FBXW7 cDNA [21], Lipofectamine and Reagent Plus (Inv-itrogen).For knockdown experiments, human FBXW7 specific [20] and negative control siRNAs were purchased from Eurogentec (Angers, France) and used at a concentration of 100 nM as previously described [43].Knockdown was verified 48 h after transfection by real-time PCR as described above. ", "section_name": "Real-time quantitative PCR (qPCR)", "section_num": null }, { "section_content": "Primary antibodies were mouse monoclonal anti-FLAG (Sigma, clone M2), anti-Aurora-A [44], anti-α-SMA (DakoCytomation, clone M0851), anti-Ki-67 (DakoCytomation, clone M7240), anti-PCNA (Santa Cruz sc-56), rabbit polyclonal anti-FLAG (Sigma), anti-Notch4 (Santa Cruz, clone H-225), goat polyclonal anti-actin (Santa Cruz, clone I-19), For western blotting, 20 μg of proteins from each tumor were separated on a 15% SDS-polyacrylamide gel, then electrotransferred onto a Hybond-S membrane (Amersham, Les Ulis, France).Membranes were blocked in PBST containing 5% skim milk for 2 h at 4°C, and incubated with primary antibodies at 1:200 dilution.After washing, immunocomplexes were identified with secondary antibodies coupled to peroxidase.The blots were visualized using chemiluminescence (ECL, Amersham -GE Healthcare Europe, Orsay, France).Immunofluorescence was carried out on 10 μm cryosections of tumors or on U87 cells grown in Labtek chambers.Tissue samples and cells were fixed in paraformaldehyde, formaline-ethanol (PCNA detection) or methanol-acetone (Aurora-A) and incubated after saturation with the primary antibodies at the concentration recommended by the supplier.After washing, slides were incubated with secondary antibodies (Alexa Fluor 488, and 546; 1:2000, Molecular Probes -Invitrogen) and mounted with Vectashield containing DAPI for nuclei staining. ", "section_name": "Western blotting and Immunohistochemistry", "section_num": null }, { "section_content": "All statistical analysis and graphs were performed using GraphPad Prism software. ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "The author(s) declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "MD performed qPCR and western blotting analysis.IA and NA performed cell transfections and immunofluorescence experiments.HL provided biopsies and all related detailed informations.CP provided the Aurora-A monoclonal antibody.MT and MABR performed and analyzed the FISH on tumor touchprints.MH performed statistical analysis.MH and SJ are the principal investigators who designed, supervised and analyzed the study; SJ edited the manuscript.All authors read and approved the final manuscript. ", "section_name": "Authors' contributions", "section_num": null } ]	[ { "section_content": "We thank Markus Welcker for FBXW7 expressing plasmids and kind advises, Frédéric Chibon for helping with BAC extraction and labeling.Part of this work was done on anonymous frozen tumors provided through the Tumor Bank of the Centre Hospitalier Universitaire de Bordeaux in compliance with institutional ethical guidelines.This work was supported by La Ligue Régionale contre le Cancer, comité de la Dordogne. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Publish with Bio Med Central and every scientist can read your work free of charge ", "section_name": "", "section_num": "" } ]
10.18632/oncotarget.5582	Efficiency of CD19 chimeric antigen receptor-modified T cells for treatment of B cell malignancies in phase I clinical trials: a meta-analysis	Chimeric antigen receptor (CAR) modified T cells targeted CD19 showed promising clinical outcomes in treatment of B cell malignances such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL) and other indolent lymphomas. However, the clinical benefit varies tremendously among different trials. This meta-analysis investigated the efficacy (response rates and survival time) of CD19-CAR T cells in refractory B cell malignances in Phase I clinical trials. We searched publications between 1991 and 2014 from PubMed and Web of Science. Pooled response rates were calculated using random-effects models. Heterogeneity was investigated by subgroup analysis and meta-regression. Fourteen clinical trials including 119 patients were eligible for response rate evaluation, 62 patients in 12 clinical trials were eligible for progression-free survival analysis. The overall pooled response rate of CD19-CAR T cells was 73% (95% confidence interval [CI]: 46-94%). Significant heterogeneity across estimates of response rates was observed (p < 0.001, I2=88.3%). ALL patients have higher response rate (93%, 95% CI: 65-100%) than CLL (62%, 95% CI: 27-93%) and lymphoma patients (36%, 95% CI: 1-83%). Meta-regression analysis identified lymphodepletion and no IL-2 administrated T cells as two key factors associated with better clinical response. Lymphodepletion and higher infused CAR T cell number were associated with better prognosis. In conclusion, this meta-analysis showed a high clinical response rate of CD19-CAR T cell-based immunotherapy in treatment of refractory B cell malignancies. Lymphodepletion and increasing number of infused CD19-CAR T cells have positive correlations with the clinical efficiency, on the contrary, IL-2 administration to T cells is not recommended.	[ { "section_content": "Immunotherapy of cancer has shown a longer time to remission and complete cures in animal studies and clinical trials [1].Adoptive immunotherapy using chimeric antigen receptor (CAR) modified T cells is a promising strategy developed in recent decades.CARs are artificial engineered receptors that can target special tumor cell surface antigen, activate T cells and further enhance T cell function MHC-independently.Objective tumor responses were reported in patients with acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and other indolent lymphomas after infusing autologous or allogeneic T cells genetically modified with CD19-CARs [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. CD19 is an antigen expressed restrictively to normal and malignant B cells but not to other normal myeloid, erythroid, megakaryocytic, or multilineage bone marrow progenitor cells [17].Therefore, CD19 is an attractive target for immunotherapies against B-cell malignancies.Compared with regular chemotherapies, application of CD19-CAR T cells showed better clinical outcomes and prognosis in treating refractory B cell malignancies.A complete remission rate of 90% and sustained remissions of up to 2 years has been reported in a clinical trial about CD19-CAR T cell therapies in patients with relapsed or refractory ALL [14].In a recent published clinical trial of CD19-CAR-T cells in treatment of CLL patients, 3 of 4 patients achieved complete remission lasted for 23 months [7].They also reported 8 of 11 lymphoma patients achieved remission lasted for 23 months.However, the clinical benefit varies broadly among different trials.In Savoldo's study, only two lymphoma patients achieved stable disease for 3 and 10 month but followed with disease progression [13].In Cruz's study, only half patients achieved completed response and partial response [3].These variations among studies might due to the different execute procedures such as the design of CAR structure, methods to introduce CAR into T cells, original T cell sources (autologous or allogeneic), T-cell culture conditions, lymphodepletion regimen, cytokine supports for T cell infusion, CAR T cell infusion schedule and dosage of CAR T cell.However, the key factor for better efficiency still remains unclear.A systematic review has examined the efficacy of CD19-CAR T cell therapies but the result was limited because only 5 clinical trails included in this review [18].In this meta-analysis, we aimed to investigate the efficiency of CD19-CAR T cells immunotherapy on current published Phase I clinical trials.We also explored the factors affected the efficiency of CAR T cells immunotherapy using meta-regression analysis. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "After duplicated literatures and meeting abstracts removed, we found 215 literatures related with CD19-CAR T cell research.Two authors identified articles eligible for further review by screen tittles and abstracts.We also found the clinical trial study literatures from the reviews.Finally, we identified 14 CD19-CAR T cellbased clinical trials for further review and analysis (Figure 1). ", "section_name": "Basic information", "section_num": null }, { "section_content": "The CAR design and manufacturing process are summarized in Table 1.Among these 131 patients, 2 patients in one clinical trial were treated with Firstgeneration CAR T cells.Six patients in another clinical trial were given with both First-generation CAR T cells and Second-generation CAR T cells.Other patients were all administered with Second-generation CAR T cells.Eighteen patients in two clinical trials infused with allogeneic T cells derived from healthy donors, and all other patients infused with autologous T cells.Electroporation, lentivirus, gammaretrovirus and retroviruses are used to introduce CAR constructs into T cells.Most of the patients received lymphodepletion before CAR T cell infusion except 32 patients.There were 3 clinical trials didn't administrate lymphodepletion to all the patients.IL-2 was admitted to T cell culture or patients as lymphodepletion regimen.There are 72 patients without any IL-2 treatment in T cell culture or lymphodepletion regimens.The medians (range) of total infused T cell number and CAR + T cell number were 2.4 ×10 8 (8.76×10 6 -1.92×10 10 ) and 1.5×10 8 (1.4×10 7 -1.1×10 10 ).(Data in cells/kg or cell/m 2 were multiplied by 60kg or 1.73m 2 respectively.) ", "section_name": "Clinical treatment strategy", "section_num": null }, { "section_content": "The response rate of CD19-CAR T cells in each clinical trial varied widely, from 0.0% [9] to 100.0% [4,6,12,[14][15][16]. Figure 2 shows the overall estimate of response rate and 95% confidence interval (CI) from the individual studies.Meta-analysis of all 14 studies yielded an overall pooled response rate of 73% (95% CI: 46-94%), with substantial heterogeneity observed (I 2 = 83.3, 2 = 77.63,P < 0.0001). ", "section_name": "Meta-analysis of response rate of CD19-CAR T cell in patients with refractory B cell malignancies", "section_num": null }, { "section_content": "", "section_name": "Table 2: Univariate and multivariable meta-regression analysis", "section_num": null }, { "section_content": "Both Begg's and Egger's regression asymmetry test showed no evidence of substantial publication bias (P = 0.260 for Begg's test; P = 0.102 for Egger's test).Then meta-regression analysis was performed based on CAR T cell protocols including T cell origin, T cell culture time, IL-2 administration to T cell culture, lymphodepletion before T cell infusion, IL-2 administration to patients, infused CAR T cell number and CAR T cell persistence time.Univariate meta-regression analysis showed that lymphodepletion, no IL-2 administration to T cells and T cell persistence more than 2 months positively associated with CD19-CAR T cells clinical responses (Table 2).Multivariable meta-regression analyses showed that lymphodepletion (P = 0.017) and no IL-2 administration to T cells (P = 0.017) were associated with heterogeneity. To confirm the results of the meta-regression, subgroups were analyzed.Firstly, we compared the clinical responses among different malignancies type (ALL, CLL and lymphoma).ALL patients have higher response rate (93%, 95% CI: 65 -100%) than CLL patients (62%, 95% CI: 27-93%) and lymphoma patients (36%, 95% CI: 1-83%) (Figure 3).Patients received no IL-2 administrated T cells had higher response rate (98%, 95% CI: 81-100%) than those received IL-2 administrated T cell (43%, 95% CI: 10-79%) (Figure 4).Patients received lymphodepletion regimen had higher response rate (88%, 95% CI: 60-100%) than patients without lymphodepletion regimen (32%, 95% CI: 1-74%) (Figure 5).Results of other non-significant difference subgroups analysis were shown in supplemental figures (Figure s1-s5) and all the detailed data were list in Supplemental Table1. ", "section_name": "Sources of heterogeneity", "section_num": null }, { "section_content": "The 6-month and 1-year PFS for total 62 patients were 80.0% and 76.3% respectively (Figure 6A).The median interval of PFS was 7.0 months.Only lymphodepletion and infused CAR + T cell number were associated with better prognosis (Figure 6B, 6C).The 6-month PFS for patients administrated with lymphodepletion regimen before T cell infusion was 94.6%, significantly higher than 54.5% in patients without lymphodepletion (P < 0.001).The 6-month PFS for patients infused more than 10 8 CAR + T cell was 94.4%, significantly higher than 58.6% in patients infused less than 10 8 CAR + T cell ( P < 0.001).The survival curves of other factors were list in Supplemental Figure 6. Cox proportional hazards regression model showed that lymphodepletion was independently associated with better prognosis (Table 3). ", "section_name": "Patient prognosis", "section_num": null }, { "section_content": "The response rates of CD19-CAR T cell in refractory B cell malignances varied widely.In current meta-analysis, the overall pooled response rate of CD19-CAR T cells in refractory B cell malignancies was 73% (95% CI: 46-94%).We also showed that lymphodepletion, no IL-2 administration to T cells and T cell persistence more than 2 months positively associated with CD19-CAR T cells immunotherapy clinical response. CD19-CAR T cells have shown effective outcomes in B cell malignancies.Compared with the regular chemotherapies which is lower than 40% response rate [19][20][21], CD19-CAR T cell immunotherapy was competitive for treatment of patients with refractory B cell malignancies.The response rate of CD19-CAR T cell varied in different B cell malignancies, with higher response rate in ALL than CLL or lymphomas.The lower response rate in CLL and lymphomas might due to the host T-cell defects, and the strong inhibitory effects from tumor microenvironment [1,[22][23][24][25].In two recent published clinical trials most of the ALL patients were adolescents aged less than 25 years old [8,14].Compared with regular regimen for adult ALL patients, adolescents and young adults could achieve better outcome from pediatric regimen [26].However, whether ALL patients with younger age benefit more from CD19-CAR T cell needs further investigation.CAR T cell immunotherapy is a multiple-step clinical practice with strict quality control clinical process.Lymphodepletion was administrated before the T cell infusion in most of the trials.Our metaanalysis showed that patients received lymphodepletion regimen had higher response rate than patients without lymphodepletion regimen.Multivariable meta-regression analyses also showed lymphodepletion was associated with higher response rates.Moreover, the survival analysis showed that patients received lymphodepletion before T cell infused had better prognosis than patients without lymphodepletion (P < 0.001).All these results suggested that lymphodepletion was a critical factor for better clinical outcomes.Lymphodepletion before T cell infusion aims to remove suppressor regulatory T cells, eliminate some cytokines dependently lymphoid cells to extend the infused CAR T-cell persistence and expansion in vivo [18,[27][28].The meta-regression also verified CAR + T cell persistence time in vivo associated with better clinical response but not independently.Considering the response rate and microenvironments differences between ALL and CLL/lymphomas, we suggest that lymphodepletion was important for the clinical outcomes of CAR T cells immunotherapy through regulation on tumor microenvironment.However, we didn't find any difference between the five lymphodepletion regimens by meta-regression.This may due to the small number of trials involved in the meta-analysis and too many variations in the regression setting.With more precious clinical trials about CD19-CAR T for B cell malignancies, the detailed association between lymphodepletion and clinical outcomes will be elaborated in the future. It is recommended that cytokines can be useful in improving the expansion of First generation CAR, and potentially benefit on Second/Third generation CAR T cells.As cytokine support, IL-2 can promote T cell expansion in vitro to improve treatment outcome in CD19-CAR T cell immunotherapy [29].But our findings showed that no IL-2 administration to T cells associated with better clinical response.Compared with Firstgeneration CARs with only a CD3 intracellular signaling domain, Second-generation CARs include another single costimulatory domain derived from either CD28 or 4-1BB [1,30].Second-generation of CARs showed superior outcomes in both animal study and clinical trials [1,30].First generation of CARs failed to elicit robust cytokine response, including IL-2 can support T cell expansion upon repeated exposure to antigen [30].The key advantage of Second generation CARs was the induction of IL-2 secretion and T cell proliferation upon CAR cross-linking [30].Among the fourteen trials in this Meta-analysis, thirteen clinical tirals used the Second generation CAR T cells, and part of them didn't use IL-2 during CAR T cell culture.In these clinical trials without IL-2 administration to T cells, anti-CD3/anti-CD28 mAb-coated magnetic beads were used to stimulate T cell expansion.CD28 stimulation may play a positive role for CAR T cell proliferation.For proliferation, CD3 antibody can provide an initial activation signal, but proliferation is dependent on co-stimulatory CD28 [31].Both CD4 + and CD8 + T cells contribute to the in vivo expansion of CAR + T cells [13].CD4 + T cells respond well to CD3/CD28 stimulation.Moreover, IL-2 might limit clonally expansion and the accumulation of antigen-specific effector T cells by promoting activation-induced cell death [32][33], but CD3/CD28 can increase T cell proliferation without provoking early cell death [34].The difference between patients received IL-2 administrated T cell and no IL-2 administration T cells may come from either IL-2 or anti-CD28.However, the difference between IL-2 stimulation and CD3/CD28 stimulation still needs more verification.There was no difference between patients administrated with IL-2 or not in lymphodepletion regimen. On-target/off-tumor effect and cytokine-released syndrome (CRS) are two major safety concerns for CAR T cell immunotherapy.On-target but off-tumor effects result from the immune response in normal cells with the CAR-targeted antigens.B-cell aplasia is an on-target but off-tumor effect of CD19-CAR-directed therapies [10][11].CRS can be caused by cytokine secretion in response to the activation of CAR T cells.CRS is often accompanied by macrophage activation syndrome, which is characteristic of hyperinflammation with prolonged fever, hepatosplenomegaly, and cytopenias [4].Among these trials involved in this meta-analysis, the infusions were well tolerated without any immediate adverse side effects in two trials [3,13].But grade 3 and grade 4 severe adverse effects associated with CD19-CAR T cell infusion were reported in all the other twelve trials, much of the toxicity that occurred in these patients was because the elevations in inflammatory cytokines.The adverse effects, fever, rigors, and dyspnea were commom within the first 24 hours, but can be controlled either by reducing the dose or between IL-6 blocker tocilizumab treatment.On-target/ off-tumor effect and B cell aplasia, was reported in six trials.However, lymphodepletion, total number of CAR T cells and T cell persistence might also correlate with the toxicities, but in this study we didn't evaluated the potential factors associated with toxicities. To improve efficiency and reduce the toxicities, several new strategies are recruited for CAR T vectors.Inducible Caspase 9 (iCasp9) was integrated to CAR construction as \"safety switch\" to control the on-target/ off-tumor toxicities [35].The combination of CAR and a second chimeric costimulatory receptor can increase the tumor target specific and avoid side effects [36].Modification CAR T cells to secrete IL-12 or using native virus-specific T cells to transducer with CAR vectors can exhibit longer persistence time of CD19-CAR T cells in microenvironment in order to enhance the efficiency [37][38].CTLA-4 and PD-1 are two important immune checkpoints negatively regulating T cell activation.Blockade PD-1, PD-L1 or CTLA-4 can prolong the efficiency of activated T cell during immune reaction [39][40].Combination of CD19-CAR T cells with PD-1/PD-1 or CLTA-4 antibodies has the possibility for better clinical outcomes. In conclusion, our meta-analysis showed a high clinical response rate of CD19-CAR T cells in refractory B cell malignancies than regular chemotherapies.The meta-analysis also found lymphodepletion regimen as a key factor associated with better clinical responses.Lymphodepletion is recommended to clinical procedures in treatment of B cell malignancies using CD19-CAR T cell-based immunotherapy.We believe that combined the new technology development with the lessons from retrospective studies would lead to better clinical efficacy by applying CD19-CAR T cells in treating B cell malignancies.www.impactjournals.com/oncotarget ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "We searched for articles published from Jan 1, 1991, to December 31, 2014 with key words \"chimeric antigen receptor\" combined by \"AND\" with \"CD19\" in both MEDLINE and Web of Science.Two authors (TF Z and CL) identified articles eligible for further review by screening tittles and abstracts.When a study was considered relevant, the article was reviewed thoroughly.Only literatures published in English and reported clinical trials with the application of CD19-CAR T cells in treatment of refractory B cell malignancies including B-ALL, B-CLL and lymphoma were eligible for further review. ", "section_name": "Search strategy and study eligibility criteria", "section_num": null }, { "section_content": "Gender, age, malignancies type, CAR design, gene transduction method, original T cell sources, T cell culture time, lymphodepletion, IL-2 administration for T cell culture and patient lymphodepletion regimen, total infused CAR + T cell number, CAR + T cell persistence time, patients' response to CAR T cells and follow-up time were all collected from each study.The primary endpoint was the response to CAR T cells immunotherapy.Response was based on the cytologic immunological test or computed tomography scans reported by each trial.Patients died not because of malignancies, lost follow-up, and with no objective disease response were excluded for analysis.Patients with response to CAR T cells immunotherapy were divided to two group: positive response group (patients achieved complete response (CR) or partial response (PR)), and negative response group (patients achieved stable disease (SD), progress disease (PD)).The response rate was calculated by the percentage of patients achieved complete response and partial response.For detailed analysis, IL-2 administration and lymphodepletion were analyzed by \"Yes\" and \"No\"; T cell origin was analyzed by \"Autologous\" and \"Allogeneic\"; T cell culture time was analyzed by \"≥ 2 weeks\" and \"< 2 weeks\"; Total T cell culture time was analyzed by \"cells > 10 8 \" and \"cells <10 8 \"; T cell persistence time was analyzed by \"≥ 2 months\" and \"< 2 months\".We assessed studies for quality on the basis of the Cochrane Collaboration's method for non-randomized studies [41]. ", "section_name": "Literature screening", "section_num": null }, { "section_content": "Metaprop is a statistical program implemented to perform meta-analyses of proportions in Stata13.0 (Stata Corp, College Station, TX) [42].Metaprop implements procedures which are specific to binomial data allows computation of exact binomial and score test-based confidence interval.It provides appropriate methods for dealing with proportions close to 0 or 100%.By using Metaprop, no studies with 0% or 100% proportions were excluded from the meta-analysis.The Freeman-Tukey double arcsine transformation was used to compute the weighted pooled response rate. We used the Cochran's Q test to assess betweenstudy differences and the I 2 statistic to quantify the proportion of observed inconsistency across study results not explained by chance.If the heterogeneity among trials were very large (I 2 statistic>75%), the observed difference between the response rates cannot be entirely attributed to sampling error and other factors such as differences in study population, etc. could also contribute.Thus, a random effects meta-analysis was used to pool the response rates of CD19-CAR T cells in refractory B cell malignances in Phase I clinical trials.The pooled response rate describes the mean of the distribution of the estimated response rate. Univariate meta-regression analyses were conducted to identify clinical factors associated with response rate.Next, we performed a multivariate meta-regression analysis on the individually significant factors from the univariate analysis.Potential interaction was also tested between potential predictors. To study possible publication bias, we evaluated Contour-enhanced funnel plots.A deficiency in the base of the funnel with asymmetry indicates the presence of possible publication bias from unpublished small studies.On a contour-enhanced funnel plot, contours of statistical significance are overlaid on the funnel plot.Publication bias was also assessed by two formal tests: Begg's adjusted-rank correlation test and Egger's regression asymmetry test. All the factors analyzed in univariate metaregression analyses were evaluated for CD19 CAR T cell immunotherapy prognosis according to progressionfree survival (PFS).The interval for PFS was defined as the time from CAR T cell infusion to disease progress.PFS curve was estimated by Kaplan-Meier method and compared by log-rank test between each factors analyzed in univariate meta-regression analyses.The Cox proportional hazards regression model was used to identify independent prognostic factors.A two-sided P value was considered as statistically significant. ", "section_name": "Statistic analysis", "section_num": null }, { "section_content": "", "section_name": "GRANT SUPPORT", "section_num": null } ]	[ { "section_content": "www.impactjournals.com/oncotargetProvince (Grant No. 112300410153, and 122300410155), Funds for Creative Research Team of Henan Province, Creative Research Team of Higher Education of Henan Province. ", "section_name": "", "section_num": "" }, { "section_content": "The authors have declared that no conflict of interest exists. This paper has been accepted based in part on peerreview conducted by another journal and the authors' response and revisions as well as expedited peer-review in Oncotarget. ", "section_name": "CONFLICTS OF INTEREST", "section_num": null }, { "section_content": "The authors have declared that no conflict of interest exists. ", "section_name": "CONFLICTS OF INTEREST", "section_num": null }, { "section_content": "This paper has been accepted based in part on peerreview conducted by another journal and the authors' response and revisions as well as expedited peer-review in Oncotarget. ", "section_name": "Editorial note", "section_num": null } ]
10.3390/cells13060557	MiR-23b and miR-133 Cotarget TGFβ2/NOTCH1 in Sheep Dermal Fibroblasts, Affecting Hair Follicle Development	<jats:p>Wool is produced and controlled by hair follicles (HFs). However, little is known about the mechanisms involved in HF development and regulation. Sheep dermal fibroblasts (SDFs) play a key role in the initial stage of HF development. Analyzing the molecular mechanism that regulates early HF development in superfine wool sheep is of great importance for better understanding the HF morphogenesis process and for the breeding of fine wool sheep. Here, we show that two microRNAs (miRNAs) affect the development of HFs by targeting two genes that are expressed by SDFs. Meanwhile, the overexpression and inhibition of oar-miR-23b and oar-miR-133 in SDFs cells and cell proliferation, apoptosis, and migration were further detected using a CCK-8 assay, an Annexin V-FITC assay, a Transwell assay, and flow cytometry. We found that oar-miR-23b, oar-miR-133, and their cotarget genes TGFβ2 and NOTCH1 were differentially expressed during the six stages of HF development in superfine wool sheep. Oar-miR-23b and oar-miR-133 inhibited the proliferation and migration of SDFs and promoted the apoptosis of SDFs through TGFβ2 and NOTCH1. oar-miR-23b and oar-miR-133 inhibited the proliferation and migration of SDFs by jointly targeting TGFβ2 and NOTCH1, thereby inhibiting the development of superfine wool HFs. Our research provides a molecular marker that can be used to guide the breeding of ultrafine wool sheep.</jats:p>	[ { "section_content": "Wool, which is a pure natural textile raw fiber material, has a high economic value and is increasingly favored by modern textile enterprises and consumers.The output of domestic fine wool, especially high-quality superfine wool, is far from meeting the processing needs of the textile market.Subo Merino is a kind of wool sheep that is independently bred in China, and its wool has a fineness of 17-19 µm.This is a new breed of sheep that produces worsted superfine wool.Improving the yield and quality of wool has always been a focus of research related to fine wool sheep breeding.Wool is produced by hair follicles (HFs), which are complex organs located in skin tissues.The first stage of HF development is the proliferation of epidermal cells to form a placode; under this structure, dermal cells accumulate, and the two cell types grow together downwards, towards the dermis, to form a dermal condensate [1].Gradually, dermal cells enter the epithelial bud to form the anterior papilla, and, finally, as the HF lengthens and enters the dermis, epithelial glomerulus cells envelop the anterior papilla [1].The first HFs that Cells 2024, 13, 557 2 of 21 are formed are primary follicles, followed by secondary follicles and then the secondaryderived follicles which branch from the secondary follicles [2][3][4].Sheep primary follicles develop mainly during the embryonic stage and complete their development process and reach maturity before birth; thus, no new primary follicles are produced at birth [5].Studies have shown that primary follicles begin to develop on day 65 of the embryonic period (E65), secondary follicles begin to form on E85, secondary-derived follicles form on E105, and HFs are basically mature on E135 [2].Mature HFs begin to cycle through the process of hair growth (anagen), catagen, and telogen [6,7].HF morphogenesis is an extremely complex biological process that is regulated by many signaling pathways [8][9][10][11] (e.g., the TGF-β, Notch, Hippo, and Wnt signaling pathways).The addition of Wnt signaling in dermal papilla cells is considered a key factor in stimulating hair growth.Mesenchymal stem cell-derived signaling and the growth factors obtained by platelets influence hair growth through cellular proliferation to prolong the anagen phase (FGF-7), induce cell growth (ERK activation), stimulate hair follicle development (β-catenin), and suppress apoptotic cues (Bcl-2 release and Akt activation) [9].It has been shown that TGF-β2 signaling is necessary to transiently induce the transcription factor Snail and activate the Ras-mitogenactivated protein kinase (MAPK) pathway in the bud.Rendl et al. identified BMP signaling as a determinant of stem cell-activating hair-inducing cell fate [12].It is also affected by epigenetics [13] (e.g., DNA/RNA methylation) and noncoding RNAs [2] (e.g., lncRNAs, circRNAs, miRNA), especially the miRNA. miRNAs are noncoding RNAs that are 22 nucleotides (nt) long and play a role in negatively regulating the post-transcriptional modification of messenger ribonucleic acids (mRNAs) in various biological processes [14].An increasing number of studies have confirmed that miRNA molecules are involved in a variety of biological processes, including growth and development, cell differentiation, apoptosis, lipid metabolism, hormone secretion, signal transduction, and stress responses [15].The dysregulation of these processes can lead to hair loss or skin problems that affect a person's appearance in mild cases and are life-threatening in severe cases.miRNAs are closely related to our lives and are worthy of further study.While much is known about the general biological functions of miRNAs, such as their interactions with the RNA-induced silencing complex, many important questions remain unanswered, especially those regarding their functions in HFs.miRNAs can regulate the expression of HF development-related genes, thereby changing the phenotype and fate of epithelial cells, fibroblasts, dermal papilla cells, and HF stem cells.HF development and morphological changes are mainly controlled by these cells [16][17][18][19].Some studies have also shown that miR-21 [20], miR-31 [21], and miR-214 [22,23] play important roles in keratinocytes.It had also been found that miR-21 affects HF development in superfine wool sheep and small-tailed Han wool sheep [24].Previous studies have shown that miR-218-5p passes the Wnt/β-catenin signaling pathway and targets SFRP2 to regulate the development of skin and HFs [19].In terms of research on the role of miRNAs in HF development, very in-depth research has been conducted on cashmere goats.CircRNA-1926 enhances the expression of CDK19 by sponging miR-148a/b-3p and promotes the differentiation of secondary HF stem cells into HF lines in cashmere goats [25].Lv et al. [26] revealed that miR-148a and miR-10a can inhibit the proliferation of Hu wool papilla cells and are related to the growth and development of HFs.miR-195-5P regulates the ability of dermal papilla cells to induce HF formation by inhibiting Wnt/β-catenin activation [27].In the HF cycle of Liaoning cashmere goats, miR-let-7a was found to regulate the expression of the C-myc, FGF5, and IGF1R genes, thereby affecting the development of HFs in said goats [28].Studies have also shown that miR-149 [29], miR-205 [30,31], miR-125b [18,32], miR-214 [22,23], and miR-218 [17] all affect the differentiation and development of HF stem cells.Interestingly, some studies have shown that miR-128 regulates the differentiation of HF mesenchymal stem cells into smooth muscle cells by targeting the major transcription regulator of TGF-β, namely, Smad2 [17]. HFs are a source of mixed cell populations because they contain cells from the epidermis and the dermis.During embryonic development, a series of mutually coordinated signals between epithelial cells from the epidermis and mesenchymal cells from the dermis trigger the formation of HFs [33,34].Dermal fibroblasts (DFs) are mesenchymal cells that are found between the skin epidermis and subcutaneous tissues [35][36][37].The mesenchymal cells of the dermis play a vital role in the formation of DFs in fetal HFs, and they play an equally important role in regulating their cyclic growth, rest, and regression phases in adults [38,39].Even within a single tissue, fibroblasts exhibit remarkable functional diversity.Fibroblasts engage in fibroblast-epidermal interactions during hair development and in interfollicular regions of the skin [40].The fibroblasts of skin connective tissue are derived from two distinct lineages [40][41][42].The developing dermis undergoes fate restriction: the cells in the upper dermis give rise to the dermal papilla, the arrector pili muscle, and papillary fibroblasts, while the cells in the lower dermis give rise to the reticular dermis and the subdermal and adipocyte layers.The upper dermis is required for HF formation [41,42].Fibroblasts play a crucial role in cutaneous wound repair.This also explains why trauma is associated with the formation of extracellular matrix component-rich scar tissues that lack HFs [43][44][45].Sustained activation of the canonical Wnt pathway in the adult epidermis induces the growth of existing HFs (anagen), the formation of new HFs [46], the proliferation of fibroblasts, and the remodeling of the dermal extracellular matrix [47,48].Since epidermal Wnt signaling promotes HF growth, the expansion of the lower dermis may explain why the skin adipocyte layer increases in thickness during aging.Dermal remodeling in response to epidermal β-catenin activation results in the expansion of the upper and lower dermis, and the increase in the upper papillary dermis allows the formation of new HFs [41,42].At present, there are few reports on the effects of miRNAs on sheep dermal fibroblasts (SDFs). Wool is an important textile material that is produced by HFs.During the development of HFs in ultrafine wool sheep, the process of embryonic HF development determines the yield and quality of the wool that will be produced by the sheep in adulthood.For example, the diameter, curvature, and density of wool fibers are related to the size of the wool substrate and the number of secondary hair follicles, and these characteristics have a high economic value in the wool industry.The structuring, functioning, and morphogenesis of HFs are complex biological processes, and the mechanism by which HF morphogenesis affects the traits of the wool that is produced by superfine wool sheep needs to be further studied.Therefore, analyzing the molecular mechanism that regulates the early development of HFs in superfine wool sheep is important for better understanding the HF morphogenesis process and for breeding sheep for wool-specific traits.Based on our previous research, we selected miRNAs (oar-miR-23b and oar-miR-133) [49] that are related to the induction of HF differentiation and key genes (TGFβ2 and NOTCH1) [50] that are related to HF development for further exploration in this study.We knocked out and overexpressed oar-miR-23b and oar-miR-133 in SDFs and used bioinformatics, Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China), reverse transcription quantitative realtime PCR (RT-qPCR), Western blot, and flow cytometry to determine the effects of miRNAs on cell proliferation, apoptosis, migration, and cycle progression.Similarly, we also tested and analyzed miRNAs that target TGFβ2 and NOTCH1.We hoped to provide a molecular theoretical basis for understanding and revealing the genetic mechanism regulating fine wool follicle development. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "", "section_name": "Materials and Methods", "section_num": "2." }, { "section_content": "Subo Merino sheep is a subtype of the Merino breed of sheep in China that is known for its high survival rate and excellent quality and yield of wool.A sheep herd located in the Kechuang Animal Husbandry Breeding Center, Xinjiang, China, was selected for testing.From this flock, twenty healthy ewes were artificially inseminated with fresh sperm from the same ram.Insemination occurred on embryonic day 0 (E0).Previous reports have described the methods used for embryonic skin tissue collection on E65, E85, E105, and E135 [49,50].The method for the collection of skin tissues on postnatal days 7 and 30 (D7 and D30) has been described previously [49,50].For each of the six developmental stages represented by the groups, three biological replicates were generated.All eighteen skin tissue samples were stored at -80 • C. Our previous article describes the details of the DE-miRNA sequence analysis [49]. ", "section_name": "Animal Selection and Skin Tissue Preparation", "section_num": "2.1." }, { "section_content": "Primary sheep dermal fibroblasts were cultured as in a previous study [49].SDFs were prepared from one newborn lamb on postnatal day 7 (local anesthesia before surgery).Surface hair was removed with a blade, and the skin was wiped with cotton balls soaked in 75% alcohol.Then, Lidocaine aerosol (Lishuka, A1004157, Shanghai Sine Pharmaceutical Laboratories Co., Ltd., Shanghai, China) was used to spray the exposed skin surface, and, after waiting for 2 min, 2 cm 2 of skin tissue was collected with a scalpel and scissors.Then, 100 IU/mL penicillin and 0.1 mg/mL streptomycin (2:100) were added to phosphate-buffered saline (PBS) at 4 • C. Briefly, the skin tissues were incubated in 0.25% trypsin at 4 • C overnight.The tissue blocks were cut into 0.8 mm 2 tissue blocks with ophthalmic scissors, broken up with a pipette, and placed in a Petri dish that contained sterile medium (Dulbecco's modified Eagle's medium, DMEM) (Invitrogen, Carlsbad, CA, USA), basal medium supplemented with double antibodies, and 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA).The cells were incubated at 37 • C with 5% CO 2 , and then the medium was changed every three days to observe cell growth in the culture dish.If fibroblasts were freed, the tissue blocks could be washed away.Subsequently, adherent fibroblasts were reseeded in new culture plates with EMEM supplemented with 10% FBS, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin.The cells were passaged when cell confluence reached a value of more than 80%. HEK-293T cells were provided by the Key Laboratory of Livestock and Poultry Multiomics of MARA (Ministry of Agriculture and Rural Affairs).HEK-293T cells were cultured at 37 • C in DMEM (Invitrogen, Carlsbad, CA, USA), supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), 1.5 mM l-glutamine (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA), and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), in a humidified incubator in an atmosphere containing 5% CO 2 (Thermo, Waltham, MA, USA).None of the cell lines used in this paper were listed in the database of commonly misidentified cell lines that is maintained by the ICLAC.All the cell lines were free of mycoplasma contamination.Adherent cells were passaged daily with 0.05% trypsin-EDTA (Invitrogen, Carlsbad, CA, USA). ", "section_name": "Cell Culture and Transfection", "section_num": "2.2." }, { "section_content": "Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).Poly-A tails were added to the miRNAs according to the protocol of the Poly (A) Tailing Kit (Ambion, Austin, TX, USA).The PrimeScript TM RT Reagent Kit with gDNA Eraser (Takara, Kusatsu, Japan) and gene-specific primers or random primers were used to generate cDNA.RT-qPCR was performed with a CFX96 TM Real-Time System (Bio-Rad, Hercules, CA, USA) using SYBR ® Green (Takara, Kusatsu, Japan) and the miRcute Plus miRNA qPCR Kit (SYBR Green) (TIANGEN, Beijing, China).Glyceraldehyde phosphate dehydrogenase (GAPDH) and U6 snRNA were used as endogenous controls for mRNA and miRNA, respectively.The thermal cycling conditions used for mRNA RT-qPCR were 95 • C for 30 s, followed by 40 cycles of 95 • C for 5 s and 60 • C for 30 s.The thermal cycling conditions used for miRNA RT-qPCR were 95 • C for 15 min, followed by 45 cycles of 94 • C for 20 s and 60 • C for 34 s.The specificity of the SYBR green PCR signal was confirmed by a melting curve analysis.There were three biological and technical replicates.The comparative Ct method was used to calculate the relative expression of the target RNAs.The primers were synthesized by Shanghai Sangong Biology Co., Ltd.(Shanghai, China).The primer sequences are displayed in Tables S1 andS2.Sequence 2 -∆∆Ct indicated the change in the expression of the gene of interest between the experimental group and the control group. ", "section_name": "RT-qPCR", "section_num": "2.3." }, { "section_content": "The total protein was extracted from the SDFs using a radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China).Protein concentration was determined using a bicinchoninic acid (BCA) kit (Thermo, Waltham, MA, USA).The proteins were separated by polyacrylamide gel electrophoresis.Next, the separated proteins were transferred onto polyvinylidene fluoride membranes (Millipore, Boston, MA, USA), which were blocked in 5% BSA at room temperature for 1 h.The membranes were incubated overnight at 4 • C with the following primary antibodies (Abclonal, Wuhan, Hubei, China): anti-Notch1 rabbit mAb (A19090, 1:10,000), anti-TGFβ2 rabbit pAb (A3640, 1:10,000), anti-Cyclin D1 rabbit pAb (A11022, 1:10,000), anti-Bax rabbit pAb (A15646, 1:10,000), anti-p53 rabbit pAb (A11232, 1:10,000), anti-CDKN1A/p21CIP1 rabbit pAb (A1483, 1:10,000), and anti-GAPDH (A19056, 1:10,000).After three washes, the membranes were incubated with diluted horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (AS014, 1:10,000, Abclonal, Wuhan, Hubei, China) for 1 h at room temperature.Then, the membranes were visualized with enhanced chemiluminescence substrates (BL520A, Biosharp, Beijing, China).The ImageJ 1.48u software (Bethesda Softworks, LLC, Rockville, MD, USA) was used for the protein quantification analysis, and GAPDH was used as the internal reference. ", "section_name": "Western Blotting Analysis", "section_num": "2.4." }, { "section_content": "miR-23b and miR-133 target genes were predicted through the online websites Tar-getScan (http://www.targetscan.org,accessed on 4 March 2023), RNAhybrid (https:// bibiserv.cebitec.uni-bielefeld.de/rnahybrid/%EF%BC%89,accessed on 4 March 2023), Pic-Tar (https://pictar.mdc-berlin.de,accessed on 4 March 2023), starBase (http://starbase.sysu.edu.cn/,accessed on 4 March 2023), and RNA22. GO and KEGG functional analyses of target genes were performed using Database for Annotation, Visualization, and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/,accessed on 18 April 2023).GO is a database established by the Gene Ontology Consortium that is used for the functional annotation and classification of target genes.A KEGG functional analysis was used to perform the functional annotation and classification of pathways in the KEGG database for the target genes.The method of the KEGG pathway's functional enrichment analysis was similar to that of the GO functional enrichment analysis.Based on the false discovery rate (FDR), we determined the GO terms and metabolic pathways that were significantly associated with the gene lists.An FDR < 0.05 was applied to significant target genes associated with GO terms or KEGG pathways. ", "section_name": "Prediction and Enrichment Analysis of Target Genes", "section_num": "2.5." }, { "section_content": "Total RNA was extracted from sheep skin according to the instructions of the TRIzol kit (Invitrogen, USA).The extracted RNA was then reverse transcribed into complementary deoxyribose nucleic acid (cDNA) according to the instructions of the One StepPrimeScript ® miRNA cDNA Synthesis Kit (TaKaRa, Kusatsu, Japan).The 3 ′ UTR fragments of NOTCH1 and TGFβ2 that contained putative oar-miR-23b/oar-miR-133 binding sites were amplified from sheep genomic DNA using forward and reverse primers containing XhoI and NotI restriction sequences, respectively.The amplified fragments were cloned at the XhoI and NotI sites downstream of the CV40 promoter-driven Renilla luciferase cassette in psiCHECK2 (Promega, Madison, WI, USA).The dual-luciferase reporter vector carrying the wild-type (WT) 3 ′ UTR of NOTCH1/TGFβ2 and the 3 ′ UTR sequences with mutations (MUTs) in the miR-23b and miR-133 binding sites were separately constructed.The primers and sequences used for the construction of wild-type and mutant vectors are shown in Table S3.HEK-293T cells with good growth were selected and seeded in 24-well plates at a density of 1 × 10 4 cells/well, followed by a culture in an incubator at 37 • C with 5% CO 2 and saturated humidity.When the cells grew to approximately a 50% confluence, cell transfection was performed according to the instructions of the Lipofectamine 3000 transfection kit (Invitrogen, USA).miRNA-23b-mimic (pro-miR-23b), psiCHECK2-NOTCH1-WT1 or psiCHECK2-NOTCH1-MUT1, psiCHECK2-TGFβ2-WT1 or psiCHECK2-TGFβ2-MUT1, and miRNA-NC (negative control) were transfected into HEK-293T cells.Similarly, miR-133 mimic (pro-miR-133), psiCHECK2-NOTCH1-WT2 or psiCHECK2-NOTCH1-MUT2, psiCHECK2-TGFβ2-WT2 or psiCHECK2-TGFβ2-MUT2, and miRNA-NC (negative control) were transfected into HEK-293T cells.The medium was replaced with fresh medium 6 h after transfection.Twenty-four hours after transfection, the relative luciferase activities were determined using the Dual-Glo Luciferase Assay System (Promega, USA).The assay was performed in triplicate for three independent trials. ", "section_name": "Luciferase Reporter Assay", "section_num": "2.6." }, { "section_content": "SDFs were cultured at 37 • C in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), 1.5 mM L-glutamine (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA), and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified incubator in an atmosphere containing 5% CO 2 (Thermo, Waltham, MA, USA).When the cells grew to approximately a 50% confluence, they were transfected with 200 nM synthetic oar-miR-23b inhibitor (anti-miR-23b) and oar-miR-133 inhibitor (anti-miR-133), oar-miR-23b mimic (pro-miR-23b) and oar-miR-133 mimic (pro-miR-133), or miRNA negative controls (mimic-NC and inhibitor-NC), using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).The cells were harvested 48 h after transfection and used for further analyses. ", "section_name": "Cell Transfection", "section_num": "2.7." }, { "section_content": "A CCK-8 assay (Beyotime, Shanghai, China) was used to analyze the cell proliferation rate.Oar-miR-23b mimic, oar-miR-23b inhibitor, mimic-NC, inhibitor-NC, oar-miR-133mimic, and oar-miR-133 inhibitor were transfected.Twenty-four hours after the SDFs were transfected, they were digested with EDTA-0.25%trypsin, and 600 µL/well (approximately 2 × 10 3 cells/mL) was seeded in a 24-well plate for culture.Four time points were established for each group: 24 h, 48 h, 72 h, and 96 h.After being cultured for 24, 48, 72 h, and 96 h, 60 µL of CCK-8 solution was administered to each well.Then, after being cultured for another 2 h, the supernatants were transferred to a 96-well plate (100 µL/well), and six replicates were established for each sample.Then, the absorbance was measured at 450 nm with a microplate reader, and the cell proliferation curve was generated according to the OD values. ", "section_name": "Cell Proliferation Assay", "section_num": "2.8." }, { "section_content": "SDFs in the logarithmic phase of growth and in good growth conditions were collected, seeded in six-well plates at a concentration of 5 × 10 5 cells/well, and cultured overnight at 37 • C in a 5% CO 2 incubator.The cells were treated according to the following groups: oar-miR-23b mimic; oar-miR-23b inhibitor; mimic-NC; inhibitor-NC; oar-miR-133-mimic; and oar-miR-133 inhibitor.After 48 h of treatment, the instructions of the Annexin V-FITC/PI apoptosis detection kit were followed, and flow cytometry was used for the analysis. ", "section_name": "Cell Apoptosis Analysis by Annexin V-FITC Staining", "section_num": "2.9." }, { "section_content": "SDFs were harvested 48 h post transfection (oar-miR-23b mimic, oar-miR-23b inhibitor, mimic-NC, inhibitor-NC, oar-miR-133 mimic, or oar-miR-133 inhibitor), and the cells were counted.Transwell chambers (Corning, NY, USA) were then placed in 24-well plates.A total of 200 µL of cell suspension (3 × 10 5 cells/mL) was added to the Transwell chamber.The cells were incubated for 24 h in a humidified incubator (37 • C/5% CO 2 ).The SDFs were then fixed with cold 70% ethanol and incubated at room temperature for 1 h.Then, the cells were stained with 0.5% crystal violet dye, photographed, and counted (10 × 10, 3 pictures/group). ", "section_name": "Transwell Cell Migration Assay", "section_num": "2.10." }, { "section_content": "SDFs were harvested 48 h post transfection (oar-miR-23b mimic, oar-miR-23b inhibitor, mimic-NC, inhibitor-NC, oar-miR-133 mimic, or oar-miR-133 inhibitor).The cells were subsequently resuspended in precooled PBS and fixed overnight at 4 • C in precooled 70% ethanol.The SDFs were washed three times with PBS, and, then, the cells were centrifuged for 5 min at 1500 rpm and resuspended in 200 µL of PBS.To each sample, 10 µL of RNase (10 mg/mL) was added and then incubated at 37 • C for 30 min.Then, 10 µL of RNaseA (400 µg/mL) was added to these cells and incubated for 30 min at 4 • C in the dark.Finally, cell cycle progression was analyzed by flow cytometry. ", "section_name": "Flow Cytometry Analysis", "section_num": "2.11." }, { "section_content": "Statistical analyses were performed using the GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA).The experimental results are expressed as the mean ± standard deviation (mean ± SD).A t test was used to analyze the differences between the two groups.One-way ANOVA was performed to analyze the differences among different groups, followed by a post hoc test (least significant difference).Significant differences were denoted by * p < 0.05, and extremely significant differences were indicated by ** p < 0.01.The plots were generated using the GraphPad Prism software (version 7). ", "section_name": "Statistical Analysis", "section_num": "2.12." }, { "section_content": "", "section_name": "Results", "section_num": "3." }, { "section_content": "In our previous research, 87 differentially expressed miRNAs (DE-miRNAs) were identified by sequencing analysis [49].We found that five DE-miRNAs (miR-23b, miR-133, miR-381-5p, miR-381-3p, and miR-655-3p) were differentially expressed in the first three stages of growth (E65, E85, and E105) (Figure 1A).Combined with previous research results of the mRNA-miRNA network [49], we selected miR-23b and miR-133 for further study.To elucidate the roles of these two miRNAs in hair follicle development, we performed RT-qPCR analysis on the skin tissues that had been isolated during the six stages of hair follicle development (E65, E85, E105, E135, D7, and D30).The results showed that miR-23b, miR-133, TGFβ2, and NOTCH1 were differentially expressed in the six phases.The expression of miR-23b showed an upward trend (Figure 1B); the expression trend of miR-133 first increased and then decreased, and its highest expression peak occurred on E105 (Figure 1C).TGFβ2 expression peaked on E85 and reached its lowest point on postnatal days 7 and 30 (Figure 1D).NOTCH1 was expressed at its lowest level on E65 (Figure 1E). Cells 2024, 13, 557 7 of 21 ", "section_name": "DE-miRNA Clustering and RT-qPCR Analysis", "section_num": "3.1." }, { "section_content": "SDFs were harvested 48 h post transfection (oar-miR-23b mimic, oar-miR-23b inhibitor, mimic-NC, inhibitor-NC, oar-miR-133 mimic, or oar-miR-133 inhibitor).The cells were subsequently resuspended in precooled PBS and fixed overnight at 4 °C in precooled 70% ethanol.The SDFs were washed three times with PBS, and, then, the cells were centrifuged for 5 min at 1500 rpm and resuspended in 200 μL of PBS.To each sample, 10 μL of RNase (10 mg/mL) was added and then incubated at 37 °C for 30 min.Then, 10 μL of RNaseA (400 μg/mL) was added to these cells and incubated for 30 min at 4 °C in the dark.Finally, cell cycle progression was analyzed by flow cytometry. ", "section_name": "Flow Cytometry Analysis", "section_num": "2.11." }, { "section_content": "In our previous research, 87 differentially expressed miRNAs (DE-miRNAs) were identified by sequencing analysis [49].We found that five DE-miRNAs (miR-23b, miR-133, miR-381-5p, miR-381-3p, and miR-655-3p) were differentially expressed in the first three stages of growth (E65, E85, and E105) (Figure 1A).Combined with previous research results of the mRNA-miRNA network [49], we selected miR-23b and miR-133 for further study.To elucidate the roles of these two miRNAs in hair follicle development, we performed RT-qPCR analysis on the skin tissues that had been isolated during the six stages of hair follicle development (E65, E85, E105, E135, D7, and D30).The results showed that miR-23b, miR-133, TGFβ2, and NOTCH1 were differentially expressed in the six phases.The expression of miR-23b showed an upward trend (Figure 1B); the expression trend of miR-133 first increased and then decreased, and its highest expression peak occurred on E105 (Figure 1C).TGFβ2 expression peaked on E85 and reached its lowest point on postnatal days 7 and 30 (Figure 1D).NOTCH1 was expressed at its lowest level on E65 (Figure 1E). ", "section_name": "DE-miRNA Clustering and RT-qPCR Analysis", "section_num": "3.1." }, { "section_content": "To further analyze the functions of oar-miR-23b and oar-miR-133, in this study, a gene ontology (GO) enrichment analysis was performed on their target genes.The top 10 GO terms are shown.According to the GO analysis, we found that, in their cellular composition (CC), oar-miR-23b's and oar-miR-133's target genes were significantly enriched in the cytosol, the nucleus, the nucleoplasm, the Golgi apparatus, the cytoplasm, and glutamatergic synapses (Figures S1 andS2, p < 0.01).However, only oar-miR-23b's target genes were enriched in the neuronal cell body, dendrites, early endosomes, and the perinuclear region of the cytoplasm (Figure S1).Similarly, only the target genes of oar-miR-133 were enriched in the basolateral plasma, the membrane, the endoplasmic reticulum membrane, the cell surface, and the ubiquitin ligase complex (Figure S2).In terms of molecular functions (MFs), the target genes of both miRNAs were significantly enriched in transcriptional activator activity, RNA polymerase II transcription regulatory region sequence-specific binding, ATP binding, RNA polymerase II core promoter proximal region sequence-specific DNA binding, transcription factor activity, sequence-specific DNA binding, and metal ion binding (Figures S1 andS2, p < 0.01).Only the target genes of oar-miR-23b were enriched in DNA binding, sequence-specific DNA binding, RNA polymerase II transcription factor activity, sequence-specific DNA binding, mRNA 3 ′ -untranslated region (3 ′ UTR) binding, and SMAD binding (Figure S1).Only the target genes of oar-miR-133 were enriched in magnesium ion transmembrane transporter activity, GTPase activator activity, protein kinase binding, protein homodimerization activity, and small GTPase binding (Figure S2).We further analyzed the biological processes (BPs) and found that the target genes of oar-miR-23b and oar-miR-133 were enriched in a negative regulation of transcription from the RNA, the polymerase II promoter, the cellular response to the leukemia inhibitory factor, peptidyl-threonine phosphorylation, and peptidyl-serine phosphorylation (Figures S1 andS2, p < 0.01).The oar-miR-23b target genes were individually enriched in endocytic recycling, microtubule cytoskeleton organization, transcription, DNA templating, protein import into the nucleus, protein autophosphorylation, and positive regulation of gene expression (Figure S1).The target genes of oar-miR-133 were also enriched in the transforming growth factor beta receptor signaling pathway, the Wnt signaling pathway, the positive regulation of cytoplasmic mRNA processing body assembly, pathway-restricted SMAD protein phosphorylation, endocytosis, and intracellular signal transduction in these biological processes (Figure S2).In conclusion, the target genes of oar-miR-23b and oar-miR-133 shared 50% of their enriched GO terms, indicating that they are not only unique but also similar. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, we found that the oar-miR-23b and oar-miR-133 target genes were significantly enriched in 14 pathways (Figures S3 andS4, p < 0.01).These pathways included axon guidance, the MAPK signaling pathway, pathways in cancer, including breast and pancreatic cancer and proteoglycans, the Ras signaling pathway, the FoxO signaling pathway, the signaling pathways regulating the pluripotency of stem cells, endocytosis, the Wnt signaling pathway, the PI3K-Akt signaling pathway, the ephingolipid signaling pathway, and the phosphatidylinositol signaling system.Additionally, 16 oar-miR-23b target genes were individually enriched in pathways including focal adhesion, transcriptional misregulation in cancer, the ErbB signaling pathway, the mTOR signaling pathway, renal cell carcinoma, chronic myeloid leukemia, non-small-cell lung cancer, EGFR tyrosine kinase inhibitor resistance, microRNAs in cancer, etc. (Figure S3).Only the oar-miR-133 target genes were enriched in the regulation of the actin cytoskeleton, the oxytocin signaling pathway, the cGMP-PKG signaling pathway, the Rap1 signaling pathway, colorectal cancer, platelet activation, cholinergic synapse, and the calcium signaling pathway (Figure S4).Interestingly, the target genes of both miRNAs were significantly enriched in the MAPK, Wnt, and PI3K-Akt signaling pathways. ", "section_name": "Functional Enrichment Analysis of Oar-miR-23b and Oar-miR-133 Target Genes", "section_num": "3.2." }, { "section_content": "To assess the interaction between oar-miR-23b and oar-miR-133 and their target genes, we constructed the relevant vectors.The results of DNA sequencing indicated that the construction of the vector was successful (Figure 2A).The interaction between each target gene and oar-miR-23b and oar-miR-133 was determined with a dual-luciferase reporter validation system, and the results showed that oar-miR-23b and oar-miR-133 inhibited the expression of their target genes.There was no significant difference in the expression of NOTCH1 and TGFβ2 between the group that had been cotransfected with the mimics and the mutant sequence (MUT group) and the group that had been cotransfected with the mimics and the control sequence.However, the fluorescence signal of the group that had been cotransfected with the mimics and the wild-type sequence (WT group) was significantly lower (Figure 2B,C, p < 0.01). Cells 2024, 13, 557 9 of 21 ", "section_name": "Oar-miR-23b and Oar-miR-133 Cotarget NOTCH1/TGFβ2", "section_num": "3.3." }, { "section_content": "To assess the interaction between oar-miR-23b and oar-miR-133 and their target genes, we constructed the relevant vectors.The results of DNA sequencing indicated that the construction of the vector was successful (Figure 2A).The interaction between each target gene and oar-miR-23b and oar-miR-133 was determined with a dual-luciferase reporter validation system, and the results showed that oar-miR-23b and oar-miR-133 inhibited the expression of their target genes.There was no significant difference in the expression of NOTCH1 and TGFβ2 between the group that had been cotransfected with the mimics and the mutant sequence (MUT group) and the group that had been cotransfected with the mimics and the control sequence.However, the fluorescence signal of the group that had been cotransfected with the mimics and the wild-type sequence (WT group) was significantly lower (Figure 2B,C, p < 0.01). ", "section_name": "Oar-miR-23b and Oar-miR-133 Cotarget NOTCH1/TGFβ2", "section_num": "3.3." }, { "section_content": "To investigate the overexpression and inhibition of oar-miR-23b and oar-miR-133 in SDF cells, we utilized RT-qPCR and WB at the mRNA level and protein level.The mRNA expression levels of NOTCH1 and TGFβ2 after the transfection of oar-miR-23b and oar-miR-133 were measured by RT-qPCR.The results showed that the expression levels of NOTCH1 and TGFβ2 after oar-miR-23b overexpression were opposite to those of oar-miR-23b, and the overexpression of oar-miR-133 resulted in opposite expression patterns to those of oar- ", "section_name": "Oar-miR-23 and Oar-miR-133 Inhibit NOTCH1/TGFβ2 Expression", "section_num": "3.4." }, { "section_content": "To investigate the overexpression and inhibition of oar-miR-23b and oar-miR-133 in SDF cells, we utilized RT-qPCR and WB at the mRNA level and protein level.The mRNA expression levels of NOTCH1 and TGFβ2 after the transfection of oar-miR-23b and oar-miR-133 were measured by RT-qPCR.The results showed that the expression levels of NOTCH1 and TGFβ2 after oar-miR-23b overexpression were opposite to those of oar-miR-23b, and the overexpression of oar-miR-133 resulted in opposite expression patterns to those of oar-miR-133 (Figure 3A,B, p < 0.01).More importantly, the expression of NOTCH1 and TGFβ2 in the oar-miR-23b overexpression group was significantly downregulated compared with that in the negative control group (p < 0.01).A significant downward trend was observed after the overexpression of oar-miR-133 compared to the negative control (p < 0.01).A significant difference was observed between the inhibition group and the control group (p < 0.01).The inhibition group exhibited significantly higher expression of NOTCH1 and TGFβ2 than the control group (p < 0.01).NOTCH1 and TGFβ2 have molecular weights of 125 and 48 kDa, respectively (Figure 3C), and the expression levels of NOTCH1 and TGFβ2 in the oar-miR-23b overexpression group significantly decreased.The expression levels of both NOTCH1 and TGFβ2 were significantly reduced in the oar-miR-133 overexpression group.However, the inhibition of oar-miR-23b and oar-miR-133 significantly increased the protein expression levels of these proteins (Figure 3D,E, p < 0.01). Cells 2024, 13, 557 10 of 21 miR-133 (Figure 3A,B, p < 0.01).More importantly, the expression of NOTCH1 and TGFβ2 in the oar-miR-23b overexpression group was significantly downregulated compared with that in the negative control group (p < 0.01).A significant downward trend was observed after the overexpression of oar-miR-133 compared to the negative control (p < 0.01).A significant difference was observed between the inhibition group and the control group (p < 0.01).The inhibition group exhibited significantly higher expression of NOTCH1 and TGFβ2 than the control group (p < 0.01).NOTCH1 and TGFβ2 have molecular weights of 125 and 48 kDa, respectively (Figure 3C), and the expression levels of NOTCH1 and TGFβ2 in the oar-miR-23b overexpression group significantly decreased.The expression levels of both NOTCH1 and TGFβ2 were significantly reduced in the oar-miR-133 overexpression group.However, the inhibition of oar-miR-23b and oar-miR-133 significantly increased the protein expression levels of these proteins (Figure 3D,E, p < 0.01). ", "section_name": "Oar-miR-23 and Oar-miR-133 Inhibit NOTCH1/TGFβ2 Expression", "section_num": "3.4." }, { "section_content": "To further understand the regulatory roles of the oar-miR-23b and oar-miR-133 genes, we selected star genes from the PI3K-Akt, MAPK, TGF-β, Hippo, and WNT pathways for RT-qPCR analysis.We found that the overexpression of oar-miR-23b significantly inhibited the expression levels of DKK1, FZD3, FZD6, HOXC13, LGR4, TGFβ1, and WNT5A (p < 0.01).The inhibition of oar-miR-23b significantly inhibited the expression of the BMP2, FZD3, FGF7, and INHBA genes (p < 0.01) but significantly upregulated the expression of the SAMD5 and WNT5A genes (p < 0.01).However, both oar-miR-23b overexpression and inhibition reduced the expression of BMP2 and FZD3, and the downregulation caused by oar-miR-23b overexpression was more pronounced (Figure 4A).We also found that the overexpression of oar-miR-133 significantly reduced the expression levels of DKK1, FZD3, FZD6, TGFβ1, TGFβ3, and WNT5A (p < 0.01).The knockdown of ", "section_name": "Effects of Oar-miR-23b and Oar-miR-133 on Pathway-Related Gene Expression", "section_num": "3.5." }, { "section_content": "To further understand the regulatory roles of the oar-miR-23b and oar-miR-133 genes, we selected star genes from the PI3K-Akt, MAPK, TGF-β, Hippo, and WNT pathways for RT-qPCR analysis.We found that the overexpression of oar-miR-23b significantly inhibited the expression levels of DKK1, FZD3, FZD6, HOXC13, LGR4, TGFβ1, and WNT5A (p < 0.01).The inhibition of oar-miR-23b significantly inhibited the expression of the BMP2, FZD3, FGF7, and INHBA genes (p < 0.01) but significantly upregulated the expression of the SAMD5 and WNT5A genes (p < 0.01).However, both oar-miR-23b overexpression and inhibition reduced the expression of BMP2 and FZD3, and the downregulation caused by oar-miR-23b overexpression was more pronounced (Figure 4A).We also found that the overexpression of oar-miR-133 significantly reduced the expression levels of DKK1, FZD3, FZD6, TGFβ1, TGFβ3, and WNT5A (p < 0.01).The knockdown of oar-miR-133 significantly reduced the expression levels of FGF7 and BMP2 and promoted the expression of TGFβ1, TGFβ3, and WNT5A (Figure 4B, p < 0.01). ", "section_name": "Effects of Oar-miR-23b and Oar-miR-133 on Pathway-Related Gene Expression", "section_num": "3.5." }, { "section_content": "To investigate the effects of the overexpression/inhibition of miR-23b and miR-133 on cell proliferation and apoptosis in SDFs, a CCK-8 cell proliferation assay was performed and apoptosis was analyzed by flow cytometry.The mRNA expression levels of the transfected miR-23b and miR-133 were measured by RT-qPCR.The results showed that miR-23b and miR-133 expression in the overexpression (mimic) group was significantly higher (Figure 5A,B, p < 0.01).The transfected cells were used in subsequent experiments.The proliferation of SDFs that had been transfected with oar-miR-23b mimic/mimic-NC and oar-miR-23b inhibitor/inhibitor-NC was assessed by the CCK-8 method.Compared with the negative control group (mimic-NC), the proliferation of cells that had been transfected with the oar-miR-23b mimic was significantly decreased (p < 0.01).However, the proliferation of inhibitortreated cells that had been transfected with oar-miR-23b was significantly higher than that of inhibitor-NC-treated cells (p < 0.05).The results showed that oar-miR-23b inhibited the proliferation of SDFs (Figure 5C).The proliferation of cells that had been transfected with the oar-miR-133 mimic was significantly lower than that of the cells that had been transfected with the mimic-NC (p < 0.01).However, the proliferation of inhibitor-treated cells that had been transfected with oar-miR-133 was significantly higher than that of inhibitor-NCtreated cells (p < 0.05).The results showed that oar-miR-133 inhibited the proliferation of SDFs (Figure 5D).The apoptosis of oar-miR-23b-transfected SDFs was analyzed by flow cytometry.Compared with the control group, the treatment group showed an increased cellular apoptosis rate.Compared with mimic-NC, overexpression of endogenous oar-miR-23b increased the apoptosis rate of SDFs.The inhibition of endogenous oar-miR-23b reduced the apoptosis rate of SDFs compared with inhibitor-NC (Figure 5E).A statistical analysis showed that the overexpression of oar-miR-23b significantly (p < 0.01) promoted the apoptosis of SDFs, while the inhibition of oar-miR-23b expression significantly (p < 0.01) inhibited the apoptosis of SDFs (Figure 5F).The treatment group exhibited increased apoptosis relative to the control group.Oar-miR-133 overexpression increased the number of SDFs undergoing apoptosis.Figure 5G shows that the inhibition of oar-miR-133 could reduce SDF apoptosis.The overexpression of oar-miR-133 significantly (p < 0.01) promoted the apoptosis of SDFs, while the inhibition of oar-miR-133 expression significantly (p < 0.01) inhibited the apoptosis of SDFs (Figure 5H). ", "section_name": "Oar-miR-23b and Oar-miR-133 Enhance Apoptosis and Inhibit Proliferation of SDFs", "section_num": "3.6." }, { "section_content": "To investigate the effects of the overexpression/inhibition of miR-23b and miR-133 on cell proliferation and apoptosis in SDFs, a CCK-8 cell proliferation assay was performed and apoptosis was analyzed by flow cytometry.The mRNA expression levels of the transfected miR-23b and miR-133 were measured by RT-qPCR.The results showed that miR-23b and miR-133 expression in the overexpression (mimic) group was significantly higher (Figure 5A,B, p < 0.01).The transfected cells were used in subsequent experiments.The proliferation of SDFs that had been transfected with oar-miR-23b mimic/mimic-NC and oar-miR-23b inhibitor/inhibitor-NC was assessed by the CCK-8 method.Compared with the negative control group (mimic-NC), the proliferation of cells that had been transfected with the oar-miR-23b mimic was significantly decreased (p < 0.01).However, the proliferation of inhibitor-treated cells that had been transfected with oar-miR-23b was significantly higher than that of inhibitor-NC-treated cells (p < 0.05).The results showed that oar-miR-23b inhibited the proliferation of SDFs (Figure 5C).The proliferation of cells that had been transfected with the oar-miR-133 mimic was significantly lower than that of the cells that had been transfected with the mimic-NC (p < 0.01).However, the proliferation of inhibitor-treated cells that had been transfected with oar-miR-133 was significantly higher than that of inhibitor-NC-treated cells (p < 0.05).The results showed that oar-miR-133 inhibited the proliferation of SDFs (Figure 5D).The apoptosis of oar-miR-23btransfected SDFs was analyzed by flow cytometry.Compared with the control group, the treatment group showed an increased cellular apoptosis rate.Compared with mimic-NC, overexpression of endogenous oar-miR-23b increased the apoptosis rate of SDFs.The inhibition of endogenous oar-miR-23b reduced the apoptosis rate of SDFs compared with inhibitor-NC (Figure 5E).A statistical analysis showed that the overexpression of oar-miR-23b significantly (p < 0.01) promoted the apoptosis of SDFs, while the inhibition of oar-miR-23b expression significantly (p < 0.01) inhibited the apoptosis of SDFs (Figure 5F).The treatment group exhibited increased apoptosis relative to the control group.Oar-miR-133 overexpression increased the number of SDFs undergoing apoptosis.Figure 5G shows that the inhibition of oar-miR-133 could reduce SDF apoptosis.The overexpression of oar-miR-133 significantly (p < 0.01) promoted the apoptosis of SDFs, while the inhibition of oar-miR-133 expression significantly (p < 0.01) inhibited the apoptosis of SDFs (Figure 5H). To investigate the effects of the overexpression/inhibition of miR-23b and miR-133 on the cell cycle of SDFs, cell cycle-related protein expression was measured by Western blotting.Cyclin D1 can promote cell proliferation, CDKN1A can inhibit cell proliferation, and BAX and P53 can promote cell apoptosis.To further understand the effect of oar-miR-23b on SDFs, we performed a Western blotting analysis of cell cycle-related protein expression.The results showed that the overexpression of oar-miR-23b effectively inhibited the protein expression of Cyclin D1 but significantly promoted the protein expression of CDKN1A, BAX, and P53.Conversely, the inhibition of oar-miR-23b effectively promoted the protein expression of Cyclin D1, while the inhibition of oar-miR-23b significantly promoted the protein expression of Cyclin D1 and inhibited the protein expression of CDKN1A, BAX, and P53 (Figure 6A-E).Combining the proliferation and apoptosis results, we can conclude that oar-miR-23b inhibits cell proliferation and promotes cell apoptosis.The apoptosis of SDFs can be promoted by oar-miR-133.The overexpression of oar-miR-133 effectively inhibited Cyclin D1 expression but significantly promoted CDKN1A, BAX, and P53 expression.In contrast, inhibiting oar-miR-133 effectively promoted Cyclin D1 protein expression (Figure 6A,C).Cyclin D1 expression was significantly increased by the inhibition of oar-miR-133, whereas CDKN1A, BAX, and P53 protein expression was significantly suppressed by the inhibition of oar-miR-133 (Figure 6A,B,D,E).Based on our findings, we can conclude that oar-miR-133 inhibits cell proliferation and promotes apoptosis.To investigate the effects of the overexpression/inhibition of miR-23b and miR-133 on the cell cycle of SDFs, cell cycle-related protein expression was measured by Western blotting.Cyclin D1 can promote cell proliferation, CDKN1A can inhibit cell proliferation, and BAX and P53 can promote cell apoptosis.To further understand the effect of oar-miR-23b on SDFs, we performed a Western blotting analysis of cell cycle-related protein expression.The results showed that the overexpression of oar-miR-23b effectively inhibited the protein expression of Cyclin D1 but significantly promoted the protein expression of CDKN1A, BAX, and P53.Conversely, the inhibition of oar-miR-23b effectively promoted the protein expression of Cyclin D1, while the inhibition of oar-miR-23b significantly promoted the protein expression of Cyclin D1 and inhibited the protein expression of CDKN1A, BAX, and P53 (Figure 6A-E).Combining the proliferation and apoptosis results, we can conclude that oar-miR-23b inhibits cell proliferation and promotes cell apoptosis.The apoptosis of SDFs can be promoted by oar-miR-133.The overexpression of oar-miR-133 effectively inhibited Cyclin D1 expression but significantly promoted CDKN1A, BAX, and P53 expression.In contrast, inhibiting oar-miR-133 effectively promoted Cyclin D1 protein expression (Figure 6A,C).Cyclin D1 expression was significantly increased by the inhibition of oar-miR-133, whereas CDKN1A, BAX, and P53 protein expression was significantly suppressed by the inhibition of oar-miR-133 (Figure 6A,B,D,E).Based on our findings, we can conclude that oar-miR-133 inhibits cell proliferation and promotes apoptosis. ", "section_name": "Oar-miR-23b and Oar-miR-133 Enhance Apoptosis and Inhibit Proliferation of SDFs", "section_num": "3.6." }, { "section_content": "We examined the effects of altering oar-miR-23b and oar-miR-133 expression on the migration and cell cycle progression of SDFs.Our results show that the overexpression of oar-miR-23b and oar-miR-133 inhibited cell migration, while the inhibition of oar-miR-23b and oar-miR-133 promoted cell migration (Figure 7A-D).Both oar-miR-23b and oar-miR-133 inhibited SDF migration.Similarly, we also found that the overexpression of oar-miR-23b and oar-miR-133 significantly promoted the G1 and G2 phases of the cell cycle but significantly inhibited the S phase of the cell cycle.However, the results of inhibiting oar-miR-23b and oar-miR-133 were opposite.We concluded that the overexpression of ", "section_name": "Oar-miR-23b and Oar-miR-133 Inhibit Cell Migration and Alter Cell Cycle Progression of SDFs", "section_num": "3.7." }, { "section_content": "We examined the effects of altering oar-miR-23b and oar-miR-133 expression on the migration and cell cycle progression of SDFs.Our results show that the overexpression of oar-miR-23b and oar-miR-133 inhibited cell migration, while the inhibition of oar-miR-23b and oar-miR-133 promoted cell migration (Figure 7A-D).Both oar-miR-23b and oar-miR-133 inhibited SDF migration.Similarly, we also found that the overexpression of oar-miR-23b and oar-miR-133 significantly promoted the G1 and G2 phases of the cell cycle but significantly inhibited the S phase of the cell cycle.However, the results of inhibiting oar-miR-23b and oar-miR-133 were opposite.We concluded that the overexpression of oar-miR-23b and oar-miR-133 promoted cell cycle progression and increased the ratio of SDF cells in the G1/S phase (Figure 7E-H).In contrast, the inhibition of oar-mir-23b and oar-miR-133 resulted in a decrease in the number of cells in the G1/S phase.These findings indicate that the downregulation of oar-miR-23b and oar-miR-133 accelerates cell cycle progression in SDFs. ", "section_name": "Oar-miR-23b and Oar-miR-133 Inhibit Cell Migration and Alter Cell Cycle Progression of SDFs", "section_num": "3.7." }, { "section_content": "During HF morphogenesis, intersecting signaling networks in epithelial and mesenchymal cells control the transcription, adhesion, polarity, and motility programs in these selected cell types.The dynamic changes in the nucleus and cytoplasm that occur during this period constitute the cornerstone of organ morphogenesis.There are two main challenges to understanding the mechanisms underlying specific processes in HFs: one challenge involves sequencing the time series of the external signals involved, and the other challenge involves analyzing how developing cells that are related to HF development translate these signals into downstream cellular remodeling, proliferation, and differentiation events.Our study provides some insights into how these events are coordinated during HF formation in developing skin.Combined with our previous research results, we found that oar-miR-23b and oar-miR-133 play key roles in the induction of HF differentiation (E65~E105), and they are expressed in skin tissue during the six stages of HF development [49].We explored the roles of oar-miR-23b and oar-miR-133 and their target genes NOTCH1 and TGFβ2 in the postnatal development of SDFs.oar-miR-23b expression gradually increased in HF development and after birth and continued to be highly expressed 30 days after birth.These results show that oar-miR-23b plays a key regulatory role in the early differentiation of HFs until the maturation of HFs.Oar-miR-133 was upregulated from E65 to E105, with the highest expression on E105.This indicates that oar-miR-133 plays a key regulatory role in SD differentiation.The target genes of these two miRNAs, namely, NOTCH1 and TGFβ2, also play roles in HF development, especially in the HF-induced differentiation stage (E65-E105). In this study, we found that the overexpression of oar-miR-23b and oar-miR-133 significantly inhibited TGFβ2 and NOTCH1 expression in SDFs and inhibited the expression of genes related to the WNT, Hippo, and TGF-β signaling pathways.The overexpression of oar-miR-23b significantly inhibited the proliferation and migration of SDFs and promoted the apoptosis of SDFs.In the current literature, research on miR-23b and miR-133 has mainly focused on human diseases [51][52][53], while the impact of miR-23b and miR-133 on HF development and SDFs has not been reported.The coculture of DFs and keratinocytes modifies the activities of both cell types [54].Keratinocytes induce the expression of TGFβ2 by DFs.DFs regulate the production of laminins and type VII collagen by keratinocytes, possibly through TGF-β signaling [54].Hu et al. [55] found that the expression of miR-23b in human immortalized keratinocytes is positively correlated with the concentration and time of TGFβ1 exposure, and they proved that miR-23b accelerates the migration of human immortalized keratinocytes by downregulating the expression of TIMP3.Some studies have also shown that miR-23b-3p overexpression can enhance the expression of collagen type I, COL1A1, COL3A1, and ACTA2 in atrial fibroblasts but has no significant effect on the proliferation and migration of atrial fibroblasts [56].Skin fibrosis is a chronic debilitating feature of several skin diseases that leads to characteristic increases in dermal fibroblast proliferation and collagen deposition through the upregulation of components of the TGF-β/SMAD pathway.Andrew M et al. [57] found that miRNAs, including miR-29, miR-196a, and let-7a, as well as decreasing the transcription of miR-21, miR-23b, and miR-31, are involved in skin fibrosis.Chen et al. [58] found that, in bovine mammary epithelial cells and mouse mammary cells, miR-133a, which can specifically target TGFβ2, is most significantly downregulated in cadmium-treated bovine mammary epithelial cells, and circ08409 can regulate the proliferation, apoptosis, and inflammation of bovine mammary epithelial cells by binding to miR-133a, thereby reducing the inhibitory effect of miR-133a on TGFβ2 expression.Other studies have shown that miR-133 affects rat cardiomyocyte apoptosis through NOTCH1 [59].Similarly, some studies have shown that miR-133 inhibits renal damage in diabetic nephropathy through the MAPK/ERK signaling pathway [60].Xu et al. [61] found that the overexpression of miR-133 inhibits the growth of glioblastoma multiforme cells and increases the cellular apoptosis rate, while the knockdown of miR-133 increases the growth of cells and decreases the cellular apoptosis rate.The overexpression of miR-133 also inhibits the proliferation and migration of primary endothelial cells by targeting FGFR1 [62].These results are consistent with our study, which showed that the overexpression of oar-miR-23b and oar-miR-133 can inhibit the proliferation and migration of SDFs and promote cell apoptosis.Hair follicle development is also regulated by dermal hair papilla cells, and, in future studies, we will explore the mechanism of action of miR-23b and miR-133 further in dermal papilla cells. TGFβ2 was upregulated from E65 to E85, indicating its critical role in SF development.TGFβ2 expression continuously decreased, beginning on E105.TGF-β2 acts as a coregulator between cells and performs multiple functions, including mediating the epithelial-mesenchymal transition, regulating cell proliferation, mediating endothelial fibrosis, and affecting cell apoptosis [63,64].Sonic hedgehog and TGFβ2 signaling also play important roles in hair follicle morphogenesis, but, in the absence of Lef-1, sonic hedgehog, or TGFβ2, compared to β-catenin-deficient skin [65,66], some hairy buds will still form.TGF-β is known to promote keratinocyte exit from the cell cycle [67].However, unlike TGFβ1-deficient skin, which exhibits an extended phase of postnatal HF growth, TGFβ2-deficient skin exhibits embryonal impairment of follicle germination [65], but approximately 50% of TGFβ2-deficient shoots do not appear to be able to progress to the downward growth stage.This feature cannot be easily explained by the previously identified effects of TGF-β.miR-370-3p inhibits the proliferation and promotes the migration of epithelial cells and fibroblasts.However, it does not affect cell apoptosis.miR-370-3p inhibits the proliferation of epithelial cells and fibroblasts by targeting TGFβR2 and FGFR2, thereby improving cell migration and ultimately regulating the fate of epithelial cells and DFs to form lamellar and dermal condensates, promoting HF morphogenesis [68].TGFβ2, which is an intercellular coregulator, performs multiple functions, including mediating epithelial-mesenchymal transformation, regulating cell proliferation, mediating endothelial fibrosis, and affecting cell apoptosis [63,64].Some studies have shown that chi-miR-199a-5p can inhibit the expression of TGFβ2 in fibroblasts, and the TGFβ2 gene is the target gene of chi-miR-199a-5p [69].This is consistent with our results, according to which TGFβ2 is targeted by oar-miR-23b and oar-miR-133, which can inhibit the expression of TGFβ2 in SDFs. NOTCH1 is upregulated during HF differentiation, indicating that it is indispensable for HF development.Although the embryonic development of HFs can be achieved without NOTCH, their postnatal development requires complete Notch signaling in the hair bulb and outer root sheath (ORS) [70].In HFs, NOTCH plays two roles: NOTCH controls the switching of cell fate of HF stem cells or their progenitor cells.In the hair bulb, NOTCH controls cell differentiation to ensure the normal development of each layer of the hair stem and inner root sheath (IRS).In hairballs, NOTCH1 is expressed at both the mRNA [71] and protein [72] levels.However, it is not expressed in presumed ORS cells.In HFs, NOTCH functions via both cellular autonomous and cellular nonautonomous mechanisms, and it is involved in intercellular communication between adjacent layers.During embryonic development, NOTCH1 mRNA expression is observed in the epidermis of the Mx, mainly in the invaginated inner cells of the epidermis, but not in the mesenchymal part [71,73].During the growth period of mature HFs, NOTCH1 is expressed in the inner cells of HFs and the basal cells of ORS [71,74].The NICD (NOTCH1 intracellular domain) that is produced after NOTCH1 activation is strongly expressed in undifferentiated hair stromal cells and the cortex and keratinocytes of the hair stem, and a small number of cells are also expressed in the keratinocytes of the ORS and IRS [75,76].In growing HFs, NOTCH1 is expressed in the cuticle and dermal papilla of the IRS of the ORS [72].In this study, we found that the NOTCH1 gene was expressed in the skin of sheep during the six stages of HF development, and the highest expression was observed on E105, indicating that the gene played an important role in HF development. Oar-miR-23b and oar-miR-133 can inhibit the expression of target genes and the translation of proteins, inhibit the proliferation and migration of SDFs, and promote the apoptosis of SDFs.The DFs in the connective tissue of sheep skin are derived from two different lineages.The cells in the upper dermis produce the dermal papilla and arrector pili muscles, the cells in the lower dermis produce the subcutaneous adipocyte layer, and the upper dermis is required for HF morphogenesis.DFs are cells associated with the formation of placodes in HF morphogenesis, which form dermal condensates and ultimately develop into dermal papilla and arrector pili muscles.Among them, the dermal papilla is the key \"control center\" in hair fiber growth and later circulation, and the arrector pili muscles are the key organs ensuring the normal growth of hair fibers.Oar-miR-23b and oar-miR-133 inhibit the proliferation and migration of SDFs, which may affect the development of placode and dermal condensate, leading to a disordered HF morphology and eventually leading to the abnormal development of HF in sheep. ", "section_name": "Discussion", "section_num": "4." }, { "section_content": "In summary, oar miR-23b and oar miR-133 can inhibit the expression of TGFβ2 and NOTCH1 at the mRNA and protein levels in SDFs.Similarly, both oar-miR-23b and oar-miR-133 can inhibit the proliferation of SDFs and promote cell apoptosis while also affecting cell cycle progression and inhibiting the migration of SDFs.Both oar-miR-23b and oar-miR-133 regulate the expression of genes in the WNT, TGF-β, and Hippo signaling pathways.In short, oar-miR-23b and oar-miR-133 exert critical regulatory effects on SDFs, which, in turn, affects the occurrence of hair follicle morphology.Our research provides molecular markers and a molecular theoretical basis for revealing the genetic mechanism underlying ultrafine wool follicle development. ", "section_name": "Conclusions", "section_num": "5." } ]	[ { "section_content": "We would also like to thank the Key Research and Development Project in the Shandong province (Competitive Innovation Platform) for its funding.We appreciate the experimental instruments and equipment provided by the Institute of Livestock and Veterinary Medicine, the Shandong Academy of Agricultural Sciences, and the Key Laboratory of Livestock and Poultry Multi omics of MARA. ", "section_name": "Acknowledgments:", "section_num": null }, { "section_content": "Data Availability Statement: All the miRNA-seq data generated in this study were submitted to the NCBI SRA database under BioProject No. PRJNA705552 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA705552,accessed on 15 February 2022).All the RNA-seq data generated in this study were submitted to the NCBI SRA database under BioProject No. PRJNA705554 (https: //www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA705554, accessed on 15 February 2022).We included other relevant data in this original manuscript file and/or in the supplementary information file.Nevertheless, the corresponding author will provide additional data related to these findings upon reasonable request. ", "section_name": "", "section_num": "" }, { "section_content": "As part of the study, the Basel Declaration was followed, and the report is in accordance with the guidelines of the ARRIVE project.All the experimental protocols were approved by the Ethics Committee of the Institute of Animal Science and Veterinary Medicine of the Shandong Academy of Agricultural Sciences (approval number: SAAS-2022-G24 and SAAS2023G01), and all the tissue samples were collected in accordance with the Ethics Committee's guidelines.We obtained all the embryo samples by cesarean section after euthanizing 12 ewes (exsanguination after electric shock syncope).Immediately after euthanasia, the skin tissues were collected and used for scientific research.The skin was collected in vivo from postnatal lambs on postnatal days D7 and D30 when they lost consciousness, with a depth of approximately 2 cm 2 × 3 mm.In the following steps, the skin wounds were sprinkled with anti-inflammatory powder and carefully sutured.Until the wound had healed, our lambs were treated with special care.We did everything we could to minimize their suffering and discomfort. Informed Consent Statement: Not applicable. The authors declare that they have no competing interests. ", "section_name": "Institutional Review Board Statement:", "section_num": null }, { "section_content": "As part of the study, the Basel Declaration was followed, and the report is in accordance with the guidelines of the ARRIVE project.All the experimental protocols were approved by the Ethics Committee of the Institute of Animal Science and Veterinary Medicine of the Shandong Academy of Agricultural Sciences (approval number: SAAS-2022-G24 and SAAS2023G01), and all the tissue samples were collected in accordance with the Ethics Committee's guidelines.We obtained all the embryo samples by cesarean section after euthanizing 12 ewes (exsanguination after electric shock syncope).Immediately after euthanasia, the skin tissues were collected and used for scientific research.The skin was collected in vivo from postnatal lambs on postnatal days D7 and D30 when they lost consciousness, with a depth of approximately 2 cm 2 × 3 mm.In the following steps, the skin wounds were sprinkled with anti-inflammatory powder and carefully sutured.Until the wound had healed, our lambs were treated with special care.We did everything we could to minimize their suffering and discomfort. Informed Consent Statement: Not applicable. ", "section_name": "Institutional Review Board Statement:", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.1186/s12935-024-03437-8	MiR-155 deficiency and hypoxia results in metabolism switch in the leukemic B-cells	<jats:title>Abstract</jats:title><jats:p>Hypoxia represents one of the key factors that stimulates the growth of leukemic cells in their niche. Leukemic cells in hypoxic conditions are forced to reprogram their original transcriptome, miRNome, and metabolome. How the coupling of microRNAs (miRNAs)/mRNAs helps to maintain or progress the leukemic status is still not fully described. MiRNAs regulate practically all biological processes within cells and play a crucial role in the development/progression of leukemia. In the present study, we aimed to uncover the impact of hsa-miR-155-5p (miR-155, <jats:italic>MIR155HG</jats:italic>) on the metabolism, proliferation, and mRNA/miRNA network of human chronic lymphocytic leukemia cells (CLL) in hypoxic conditions. As a model of CLL, we used the human MEC-1 cell line where we deleted mature miR-155 with CRISPR/Cas9. We determined that miR-155 deficiency in leukemic MEC-1 cells results in lower proliferation even in hypoxic conditions in comparison to MEC-1 control cells. Additionally, in MEC-1 miR-155 deficient cells we observed decreased number of populations of cells in S phase. The miR-155 deficiency under hypoxic conditions was accompanied by an increased apoptosis. We detected a stimulatory effect of miR-155 deficiency and hypoxia at the level of gene expression, seen in significant overexpression of <jats:italic>EGLN1</jats:italic>, <jats:italic>GLUT1</jats:italic>, <jats:italic>GLUT3</jats:italic> in MEC-1 miR-155 deficient cells. MiR-155 deficiency and hypoxia resulted in increase of glucose and lactate uptake. Pyruvate, ETC and ATP were reduced. To conclude, miR-155 deficiency and hypoxia affects glucose and lactate metabolism by stimulating the expression of glucose transporters as <jats:italic>GLUT1, GLUT3</jats:italic>, and <jats:italic>EGLN1</jats:italic> [Hypoxia-inducible factor prolyl hydroxylase 2 (HIF-PH2)] genes in the MEC-1 cells. </jats:p>	[ { "section_content": "Leukemic cells, due to their high-energy demand, are forced to balance the oxygen deprivation in their original microenvironment by altering their metabolism [1].One of the main manifestations of hypoxia at the cellular level is the modulation of cell proliferation.In general, cells adapt to hypoxia by expression of hypoxia-inducible factor (HIF) proteins.The HIF family is highly sensitive to intra-tissue pO 2 and regulates more than 200 genes which reestablishes oxygen homeostasis and promotes cell survival in hypoxia [2].The key molecule of this pathway is Hypoxia Induced Factor 1α (HIF-1α) which is often upregulated in tumors due to intratumoral hypoxia or by activation of some oncogenic pathways such as PI3K, TGFβ, MYC, and NOTCH.HIF-1α is overexpressed in the early stages of hypoxia (few hours) and is associated with aggressive behavior of leukemic cells and with low survival of patients [3].Besides HIF-1α, the hypoxia-related genes (EGLN1, VHL, HK1, HK2, LDHA, VEGFA, PTEN, PIK3CA) participate in the modulation of cell proliferation and metabolism and thus maintain the leukemic status [3].CLL cells show high plasticity to hypoxic conditions that results in metabolic adaptation, mainly involving glucose and pyruvate [4]. In hypoxia, malignant cells increase their proliferation and glucose uptake through glycolysis [5].The key sensors of glycolysis during hypoxic conditions in leukemic cells are glucose transporters (especially GLUT1 and GLUT3) [6].In humans, there exists three families of genes that code glucose transporters: (1) SLC2A genes that code sodium-independent glucose transporters (facilitated transport, GLUT proteins); (2) SLC5A genes, that code sodium-dependent glucose symporters (secondary active transport, SGLT proteins), and (3) SLC50A genes, which code SWEET protein [7].The family of sodium-independent glucose transporters contains 14 members GLUT1-GLUT14, encoded by SLC2A1-SLC2A14 genes, respectively [8].Measurement of the level of glucose transporters (glucose uptake) is used as a diagnostic technique for cancers (18F-deoxyglucose positron emission tomography, FDG-PET) [9].Therefore, therapeutic inhibition of glucose transporters may be an approach for future treatments for patients with different malignancies.Small non-coding microRNAs (miRNAs) could serve as mRNA targeted inhibitors (small therapeutic inhibitory molecules).It was described that miRNA-195-5p inhibits GLUT3 resulting in the inhibition of glucose uptake and growth of bladder cancer cells [10].Similarly, inhibition of GLUT1 by miRNA-125a-5p was described in thyroid carcinoma [11].There is evidence that miRNAs are important regulators of glucose metabolism [12].Interestingly, there are only few papers describing the direct effect of miR-155 on glucose metabolism but none in the relation of miR-155 to CLL cells and hypoxia.For example, Kim et al., 2018 found that the deletion of miR-155 in breast cancer cells abolishes the glucose uptake [13]. Hypoxic cells show high levels of lactate which serves as a signaling molecule, as an end product of anaerobic glycolysis, and is involved in fatty acid synthesis and redox homeostasis [14].Lactate dehydrogenase (LDHA) catalyzes conversion of pyruvate into lactate.Future miRNA-based therapies could be used to modulate tumor/leukemic cell metabolism [15]. HypoxamiRNA (hypoxia related miRNA) significantly modulates gene expression through its targets thus improving adaptation of the leukemic cells to hypoxia [16].The most studied hypoxamiRNAs is miR-210 which is significantly overexpressed in several cancers during hypoxic conditions.Overexpression of miR-210 is associated with a poor prognosis [17].Here we focus on the oncomiRNA and possible hypoxamiRNA, miR-155 in CLL (MEC-1 cell line).MiR-155 is an essential regulatory molecule involved in multiple physiological processes, including hematopoietic lineage differentiation, immune response, and inflammation [18,19].Moreover, expression of miR-155 is directly associated with cancer progression and disease aggressiveness, including in CLL [20][21][22].MiR-155 is expressed ubiquitously in different cells thus any knowledge about its function will be valuable in every research area. Hypoxia related genes as HIF1α and VHL are direct validated targets of miR-155 [37].MiR-155 acts as an inhibitor of tumor-suppressor pVHL that controls degradation of HIF1α protein.Overexpression of miR-155 leads to HIF1α protein stability in primary CLL cells [23].Griggio et al., 2020 reported an aberration in the tumor-suppressor gene TP53 that is also associated with higher expression level of HIF1α [24] which has an unfavorable prognosis in CLL patients [25]. In this study, we tracked the role of oncogenic and possible hypoxic related miR-155 in MEC-1 cells. We aimed to answer the question whether miR-155 deficiency affects cell proliferation, cell cycle, and the gene expression profile under hypoxic versus normoxic conditions.We found that hypoxia leads to the modulation of miRNA/mRNA network and in the absence of miR-155 in MEC-1 cells impair cell proliferation, switches glucose and lactate metabolism. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Materials and methods", "section_num": null }, { "section_content": "We acquired the original MEC-1 cell line (#ACC 497, DSMZ) as a kind gift from Prof. Marek Mraz, M.D., Ph.D. (Laboratory of Microenvironment of Immune Cells, Central European Institute of Technology (CEITEC MU) and University Hospital Brno, Czech Republic).We modified the original MEC-1 cells using CRISPR/ Cas9.A CRISPR/Cas9 plasmid (U6gRNA-Cas9-2A-GFP MiR155) was introduced into MEC-1 cells by nucleofection by Amaxa Nucleofector II (Lonza) with use of the B-cell nucleofection kit (#VPA1001, Lonza) and U-015 program.The CRISPR/Cas9 plasmid we ordered from Sigma-Aldrich, was designed at our laboratory and targeted the mature hsa-miR-155-5p sequence.More information about the creation and validation of miR-155 deficient MEC-1 clone #48 are in the Supplemental Fig. 1.MEC-1 cell line was cultured in IMDM medium (#LM-I1090, Biosera) supplemented with 10% of fetal bovine serum (#FB-1090, Biosera), 1% of P/S (#P4333, Sigma Aldrich) at 37 °C in 5% CO 2 incubator.We followed the recommended protocol for cell culturing from DSMZ for MEC-1 cells (#ACC-497).The hypoxic MEC-1 cell line was incubated in hypoxic glove box (Coy O 2 Controlled InVitro Glove-Box-Hypoxia Chamber, Genetica) in IMDM medium supplemented with 10% of fetal bovine serum, 1% of P/S at 37 °C, 1% O 2 (for optimizing experiments we used 0.2%, 1% and 5% O 2 ) and 5% CO 2 for 24 h, and 48 h (for optimizing experiments also 72 h, 96 h and 120 h).Cells were collected for RNA isolation, proliferation (WST-1 assay), cell viability (AnnexinV/ PI staining), cell cycle kinetics (BrdU staining) and metabolic assays. ", "section_name": "Cell line and experimental model", "section_num": null }, { "section_content": "Chemically induced hypoxia was made in vitro through the addition of deferoxamine mesylate salt-DFO (#D9533, Sigma-Aldrich) and dimethyloxalylglycine, N-(Methoxyoxoacetyl)-glycine methyl ester -DMOG (#D3695, Sigma-Aldrich) to a well containing 2 × 10 6 cells.Cells were cultured in IMDM medium (LM-I1090, Biosera) supplemented with 10% of fetal bovine serum (#FB-1090, Biosera) and 1% of P/S (#P4333, Sigma Aldrich).Cells were then incubated for 3 h, 6 h, and 24 h at 37 °C in 5% CO 2 with DFO (final concentration 200 µM) and DMOG (final concentration 1 mM).After each time interval of incubation with DFO or DMOG, cells were collected, and total RNA was extracted for further gene expression detection.In parallel, cells were collected for flow cytometry measurement of apoptosis (AnnexinV/PI staining). ", "section_name": "Chemically induced hypoxia", "section_num": null }, { "section_content": "The WST-1 assay was used for the cell proliferation and viability measurement, followed by manufacturer's protocol (ROCHE, #11 644 807 001).MEC-1 cells were seeded 5000 cells per well (used 96 well plate, flat bottom) in culture medium and incubated with 10 µL of WST-1 solution for 3 h [in normoxia and hypoxia conditions (1% O 2 )].After 3 h, the substrate reaction absorbance was measured at 440 nm wavelength (blank 600 nm) by using Spark ® multimode microplate reader (TECAN) spectrophotometer (Tecan i-control, 1.10.4.0, infinite 200Pro).The absorbance of blank was subtracted from measured samples wavelength.Data are from three A Heat map shows changes (as Fold Change, FC) in the mRNA/miRNA expression level in different hypoxia conditions.Hypoxia conditions were set as follows: 0.2, 1 and 5% O 2 for a period of 24 h.B Heat map shows changes in the mRNA/miRNA expression level in 1% O 2 (hypoxia) in MEC-1 ctrl and miR-155 -/-cells in different time-periods (24 h-120 h).C Heat map summarizes the expression level of selected hypoxia-related genes measured in the final hypoxia and time-period settings in MEC-1 cells, 1% and 24 h-48 h, respectively.In all 3 heat maps, the expression data were normalized to the expression level measured in normoxia and set as 1.The gene expression in all heat maps is shown as fold change (FC) and its intensity expresses the color scale next to each heat map independent experiments.Data were analyzed using t-test, two-tailed, paired. Cell growth curve was created by cell count determined by hemocytometer (counting-chamber).Cells were counted daily for one-week (in parallel normoxia vs hypoxia (1% O 2 ) conditions).Seeding density of cells at day 0 was 10,000.Data are from three independent experiments (samples were done in quadruplicate).Data were analyzed using t-test, two-tailed, paired. Annexin V/PI staining was performed for evaluation of apoptotic and dead cells.MEC-1 cells (1 × 10 6 cells) were washed with 1 × PBS, resuspended in 1 × Annexin V binding buffer, stained by 5 µL of Annexin V (15 min at RT) (FITC, # BMS500FI-300, Invitrogen).Cells were washed with 1 × PBS and kept on ice until measurement on flow cytometer.Shortly before measurement 5 µL of PI (# BMS500FI-300, Invitrogen) was added.Data were evaluated by Diva software, the software FlowJo was used for data visualization, and the measurement was performed with the use of the FACS Canto II BD flow cytometer (30 000 events). ", "section_name": "Proliferation and cell viability tests", "section_num": null }, { "section_content": "For BrdU staining, 2 × 10 6 cells/well was used.A volume of 30 uL of BrdU were added to the MEC-1 cell culture (final concentration 1 µM) and stained for 20 min at 37 °C in 5% CO 2 and 21% O 2 under normoxic and under hypoxic (1% O 2 ) conditions as followed by the manufacturer's protocol (# 552598, BrdU flow kit, BD).Before flow cytometry measurement, cells were stained with 7AAD (provided by BrdU kit, 20 µL/tube) for 10 min on ice.Measurement was performed on cytometer FACS Canto BD (50 000 events).The software FlowJo was used for data visualization.Data are from three independent experiments.Data were analyzed using t-test, two-tailed, paired. ", "section_name": "Cell cycle measurement by BrdU flow kit", "section_num": null }, { "section_content": "Total RNA including microRNA was extracted from MEC-1 cells (2 × 10 6 ) by TRI reagent (#TR118, MRC) with slight modification as over night precipitation with isopropanol at -20 °C and followed by manufacturer's protocol.An amount of 100 ng of total RNA including microRNA was reverse-transcribed by High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (#4,374,966, ThermoFisherScientific). TaqMan-based PCR with specific probes (Universal Probe Library, ROCHE and ThermoFisherScientific primers designed with probes) was performed on QS7 Pro instrument (ThermoFisherScientific).As reference genes, GAPDH (for mRNA) and RNU44 (for miRNA) were used.The miRNA/mRNA expression was calculated by 2 deltadelta CT algorithm from target and reference CT values [specific (s) and control (c) amplicons calculated by 2 -(CTc-CTs) equation] [26].Data were acquired using t-test, two-tailed, paired (p < 0.05; p < 0.01; p < 0.001; **p < 0.0001). ", "section_name": "RNA isolation and qRT-PCR", "section_num": null }, { "section_content": "Cells were lysed (2 × 10 6 ) in RIPA buffer and sonicated (1 cycle, 10\" at 40% power).Four micrograms of proteins were separated by 1-D polyacrylamide gel electrophoresis (Mini-PROTEAN ® TGX ™ Precast Gels, Bio-Rad).Proteins were transferred to Immuno-Blot ® PVDF Membrane (#1,620,174, Bio-Rad) using Trans-Blot ® Turbo ™ Transfer System (Bio-Rad) and probed with primary antibody overnight at 4 °C (anti-HIF-1α, sc-10790; anti-GAPDH sc-51907, Santa Cruz Biotechnology).The following day, the membrane was washed with 1 × TBS and probed with secondary antirabbit antibody (#A0545, Sigma-Aldrich), conjugated with horseradish peroxidase and detected using detection kit (#1,705,060, Clarity ™ Western ECL Substrate, Bio-Rad).Signalling was detected using ChemiDoc MP Imaging System (Bio-Rad). ", "section_name": "Western blot", "section_num": null }, { "section_content": "Mitochondria isolation was followed by the referred protocol in [27].Mitochondria protein content was measured by the BCA kit (#BCA1-1KT, Sigma-Aldrich) on Biotek Synergy HT Microplate Reader at 562 nm wavelength.The concentration of protein was calculated using a standard curve prepared using the BSA protein standards. Electron transport chain complex I to complex III was followed protocol in [27].Mitochondria protein content was measured by the BCA kit (#BCA1-1KT, Sigma-Aldrich).Absorbance (reduction from succinate to cytochrome C) was read at 550 nm wavelength for 6 min (1 read/15 s) on Biotek Synergy HT Microplate Reader.Results were calculated according to Lambert-Beer equation. The Glucose Uptake-GloTM Assay (#J1341, Promega) was used according to the manufacturer´s manual.The MEC-1 cells were cultured in normoxia and hypoxia (1% O 2 , 24 h, and 48 h), then counted and 10,000 cells were transferred to a non-translucent 96-well cell culture plate.The reaction solution from the kit was added and the luminescence signal was acquired at 0.5 s on the Spark ® multimode microplate reader (Tecan i-control, 1.10.4.0, infinite 200Pro) and is shown as counts/s. ", "section_name": "Metabolic assays", "section_num": null }, { "section_content": "(#N13195, ThermoFisherScientific) was used for the measurement of glucose uptake in MEC-1 cells (ctrl and miR-155 deficient) as followed by the manufacturer´s recommendations.The MEC-1 cells were counted and 200,000 cells were seeded on 6-well plate (in triplicates) and then cultured in normoxia and hypoxia (1% O 2 ) for 24 h and 48 h.Cells were washed with 1 × PBS, resuspended in 1 mL of Seahorse XF DMEM medium (#103,575-100, Agilent), and then incubated 10 min with 2-DG in final concentration 10 µM (in normoxia and in hypoxia).Next, cells were washed with 1xPBS and the cell pellet was resuspended in 200 µL of 1xPBS, followed by flow cytometry.Shortly before measurement, 5 µL of PI (# BMS500FI-300, Invitrogen) were added.As 2-DG has an excitation/emission maximum of ∼465/540 nm, we used channel FITC 530 in this case.Data were evaluated by Diva software and the measurement was performed with the use of the FACS Canto II BD flow cytometer (50 000 events). The Lactate Assay (#MAK064, Sigma-Aldrich) was used according to the manufacturer´s manual.The MEC-1 cells were cultured in normoxia and hypoxia (1% O 2 , 24 h, and 48 h), were then centrifuged (350´ g, RT, 5 min), and 10 µL of supernatant were aspirated for the assay and transferred to 96-well plate.Each sample was brought to a final volume of 50 µL/well with Lactate Assay Buffer.The reaction solution from the kit was mixed and 50 µL of Master Reaction Mix were added to each sample.Cells in the 96-well plates were incubated for 30 min at RT protected from light.Colorimetric absorbance was measured on Biotek Synergy HT Microplate Reader at 570 nm wavelength.Concentration of lactate was calculated using standard curve. mtATP level was obtained by the Adenosine 5′-triphosphate (ATP) Bioluminescent Assay Kit (#FLAA, Sigma Aldrich).The MEC-1 control group and the MEC-1 miR-155 deficient cells (2 × 10 5 cells) were cultivated 24 h and 48 h under the normoxia and hypoxia (1% O 2 ) conditions.Cells were cultured under standard cell culture conditions.Mitochondria were isolated and further mtATP measurement was carried out according to the manufacturer´s protocol.Lumenescence was measured by the Biotek Synergy HT Microplate Reader. ATP level was determined by the CellTiter-Glo ® 2.0 Cell Viability Assay (#G9242 Promega).The MEC-1 ctrl and the MEC-1 miR-155 deficient cells were cultivated 24 h and 48 h under the normoxia and hypoxia (1% O 2 ) conditions.Cells were cultured under standard cell culture conditions and 5,000 cells were used for measurement.The assay was performed according to the manufacturer's manual.The luminescence signal was acquired at 0.5 s on the Spark ® multimode microplate reader (Tecan i-control, 1.10.4.0, infinite 200Pro) and is shown as counts/s. LDH level was acquired by the LDH-Glo ™ Cytotoxicity Assay (#J2380 Promega).The MEC-1 ctrl and the MEC-1 miR-155 deficient cells were cultivated 24 h and 48 h under the normoxia and hypoxia (1% O 2 ) conditions.Cells were cultured under standard cell culture conditions and 2 µL of the cultivation media out of 1 ml was used for the measurement.The assay was performed according to the manufacturer´s manual.The luminescence signal was acquired at 0.6 s on the Spark ® multimode microplate reader (Tecan i-control, 1.10.4.0, infinite 200Pro). Pyruvate concentration was determined by the Pyruvate Assay Kit (#MAK332 Sigma-Aldrich).The MEC-1 control group and the MEC-1 miR-155 deficient cells were cultivated 24 h and 48 h under the normoxia and hypoxia (1% O 2 ) conditions.Cells were cultured under standard cell culture conditions and 1 × 10 6 were homogenized mechanically in 1 ml of cold PBS; 10 µl were used for the measurement.The assay was performed according to the manufacturer´s manual.The fluorescence signal was acquired, excited by 530/20 nm and emission was read at 585/20 nm on the Spark ® multimode microplate reader (Tecan i-control, 1.10.4.0, infinite 200Pro). ", "section_name": "2-NBDG", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Due to a relatively wide interval of oxygen level, in the normal tissues between 1-11% and in the tumor about 2% [28] and lack of consistent hypoxia experimental data, we started with optimizing the oxygen level and time periods for further hypoxia experiments.First, we cultured MEC-1 cells (ctrl and miR-155 -/-) in different oxygen levels (0.2%, 1% and 5%) in the hypoxic glove box.The final oxygen level was set up by following criteria: high expression of hypoxia related genes (HIF1α, EGLN1, LDHA, VEGFA, GLUT1, GLUT3, HK1, HK2) and low expression of TP53INP1.As the highest expression level of LDHA, HK1, GLUT1, GLUT3 and the lowest expression of the TP53INP1 (the anti-proliferative and pro-apoptotic protein) were detected in the 1% O 2, this O 2 concentration was determined to be the most suitable for further experiments (Fig. 1A).Next, we optimized the time period for incubation of MEC-1 cells in 1% O 2 hypoxia.With prolonged incubation of cells in hypoxia the level of apoptosis increased (Figs. 2C and1B), so the best time period based on the expression level (low) of TP53INP1 mRNA was set up for 24 h and 48 h (Fig. 1B). Figure 1C contains the final selection of the gene expression profile detected under hypoxia (1% O 2 ) within 24 h and 48 h time periods.Our gene expression data showed extremely elevated levels of a well-known hypoxamiRNA, miR-210, in MEC-1 cells in hypoxia (1% O 2 ) (Fig. 1C).The elevated level of miR-210 served as an additional internal control of presence of hypoxia in cells.The highest mRNA level showed hypoxia-related genes such as EGLN1, GLUT1, GLUT3, VEGFA, LDHA, and HK1.The lower level of miR-155 in MEC-1 control cells is probably the result of adaptation of the cells to the hypoxic condition (Fig. 1C).The general low expression level of HIF1α (also at the protein level, Supplemental Fig. 2) could be caused by its low stability and fast degradation within hours in hypoxia [39].Another possible reason could be the high level of EGLN1 mRNA (as its primary function is to degrade HIF1α protein) and low level of VHL or another secondary effect of miRNAs regulatory pathway on the HIF family [40]. ", "section_name": "Optimization of the oxygen level and time points for in vitro culturing of MEC-1 cells in hypoxia", "section_num": null }, { "section_content": "Hypoxia in general stimulates leukemic and cancer cells proliferation.Here we asked if the deletion of miR-155, oncomiRNA and probable hypoxamiRNA impairs the proliferation of MEC-1 cells in hypoxic conditions.Usually, hypoxia stimulates the proliferation of cells.Here we observed the opposite effect in relation to miR-155 expression.This observation was in concordance with our hypothesis, where we assumed superior effect of miR-155 expression toward hypoxia in the leukemic MEC-1 cells.Here we detected that the cell proliferation significantly decreased in miR-155 deficient MEC-1 cells under both oxygen conditions, in comparison to the MEC-1 control Additionally, we conducted the statistical analysis of MEC-1 ctrl cells vs miR-155 -/-in normoxia and so on in the hypoxia for the whole Figure 2. The statistical significance is shown in the Supplemental Material/Tables.cells (Fig. 2A,B; and Supplemental Material/Tables).Decreased cell proliferation is mediated by miR-155 expression in miR-155 deficient MEC-1 cells, confirmed by proliferation curve and WST-1 assay (Fig. 2A,B).The significant difference in the cell proliferation was visible from day 4 (Fig. 2A).We also assessed the statistical difference between the MEC-1 control and MEC-1 miR-155 deficient cells in different oxygen conditions (normoxia and hypoxia separately) (Supplemental Material/Tables).Deficiency of miR-155 resulted in the increased number of apoptotic cells (Fig. 2C).Next, for assessment of the cell cycle kinetics, we labeled MEC-1 cells by BrdU.BrdU labeling points out significantly lower percentage of cells in the S phase, more distinct in miR-155 deficient MEC-1 cells (Fig. 2D, left graph).Hypoxia enriched the cell population in G0/G1 phase more prominently in miR-155 -/-MEC-1 cells (Fig. 2D, right graphs).The statistical significance is shown in Supplemental Material/Tables. In conclusion, hypoxia significantly stimulates proliferation of MEC-1 cells depending on the miR-155 expression.MiR-155 is important for cell growth, and its absence even in hypoxia results in the lower proliferation of miR-155 deficient MEC-1 cells with increased level of apoptosis in comparison to control MEC-1 cells. ", "section_name": "MiR-155 deficiency results in the lower cell proliferation", "section_num": null }, { "section_content": "We chemically-induced hypoxia with DFO and DMOG in MEC-1 cells to validate the data from the hypoxia glove box.Chemical inducers of hypoxia DFO and DMOG operate rapidly, thus we selected short time periods as 3 h, 6 h and 24 h for their assessment (Fig. 3A).We measured the same set of genes as in Fig. 1C.Expression levels of EGLN1, GLUT3 and VEGFA mRNA showed the highest fold change in both control and miR-155 deficient MEC-1 cells (Fig. 3A).The gene GLUT3 showed significantly highest expression level after 24 h in miR-155 deficient MEC-1 cells (after DFO + 13,0 FC; after DMOG + 8,0 FC) in comparison to MEC-1 control cells (after DFO + 8,0 FC; after DMOG + 4,0 FC) (Fig. 3A).Interestingly, in the hypoxia glove box the GLUT3 reached much lower expression (+ 3,0 FC in control and + 2,0 FC in miR-155 -/-, 48 h, Fig. 1C) and surprisingly the GLUT1 showed the highest expression level in miR-155 deficient MEC-1 cells (+ 5,0 FC, in 48 h, Fig. 1C).As expected, chemically-induced hypoxia significantly increased the expression of well-known hypoxi-amiRNA-210 in both control (after 24 h of DFO + 8,0 FC and of DMOG + 8,2 FC) and miR-155 deficient MEC-1 cells (after 24 h of DFO + 6,2 FC and of DMOG + 7,8 FC; Fig. 3A).Similarly, we detected upregulation of miR-210 in hypoxia performed in the hypoxia glove box; for MEC-1 control (24 h + 4,0 FC; 48 h + 5,0 FC) and for miR-155 -/-MEC-1 cells (24 h + 4,0 FC; 48 h + 6,0 FC) (Fig. 1C).The expression of miR-155 in MEC-1 control cells in both, chemically-induced hypoxia and hypoxia in the hypoxia glove box did not increase much (Fig. 1C and Fig. 3A).Both chemical inducers of hypoxia showed similar trends in the gene expression level of selected genes.As these chemicals induced the total hypoxia in cells, we measured the level of apoptosis during all selected time periods in MEC-1 cells.Based on the flow cytometry data from measurement of Annexin V/PI we could conclude that in the abovementioned conditions the chemicallyinduced hypoxia caused overall slightly increased level of apoptosis in MEC-1 control cells.Overall higher apoptosis was detected in the miR-155 deficient MEC-1 cells, with a peak after 24 h time period (~ 28% after DFO) (Fig. 3B).In summary, the chemically-induced hypoxia vs hypoxia performed in the hypoxia glove box in MEC-1 cells showed similar trend in some genes.The most significant change was detected in the expression of GLUT3.GLUT3 mRNA showed + 13,0 FC increasement after DFO and + 8,0 FC after DMOG (24 h time period) in miR-155 deficient MEC-1 cells.Also, miR-155 deficient MEC-1 cells seem to be more sensitive to apoptosis in comparison to MEC-1 control cells. ", "section_name": "Chemically-induced hypoxia stimulates hypoxia-related genes and hypoxamiR-210 in MEC-1 cells", "section_num": null }, { "section_content": "In oxygen-deprived conditions leukemic cells shift their cellular metabolism from oxidative phosphorylation to glycolysis.This results in increased glucose uptake and significant overall changes in metabolism.Here we asked whether absence of miR-155 in MEC-1 cells during hypoxia also leads to higher glucose uptake.Interestingly, miR-155 deficiency led to increase of glucose uptake in hypoxia, significant only in miR-155 deficient MEC-1 cells after 48 h (Fig. 4A).Additionally, we performed measurement of 2-deoxy-glucose (2-NBDG) by flow cytometry, and we observed a similar trend of increased glucose uptake.Data show an increased glucose uptake by MEC-1 cells in hypoxia, significantly only in MEC-1 control cells, while in the miR-155 deficient cells we did not observed much change (Fig. 4B).From our preliminary data from mRNA-seq (not shown), we know that miR-155 deficiency results in overexpression of LDHA gene that encodes the last enzyme of anaerobic glycolysis.Here we detected overexpression of LDHA mRNA by qRT-PCR (Fig. 1A,C).As expected, we found the level of lactate increased in hypoxia; this is in line with the effect of miR-155 deficiency on LDHA levels where the production of lactate increased more rapidly in miR-155 deficient MEC-1 cells (Fig. 4C), suggesting an enhanced lactate metabolism.We also measured the level of lactate dehydrogenase (LDH) by luminescence assay, which appeared to have a tendency to rise in hypoxia (Fig. 4D).On the contrary, the level of pyruvate during hypoxia decreased (Fig. 4E).While the mitochondria represents a key organelle of energetic metabolism, their efficiency is turned off in hypoxia [38].Here we additionally measured the activity of Electron Transport Chain (ETC) and mitochondrial (mt)ATP and ATP in MEC-1 cells under hypoxia.We observed that hypoxic MEC-1 cells had reduced ETC (Fig. 4F), mtATP (Fig. 4G) and ATP (Fig. 4H).Notably, the decrease in these parameters was strikingly higher in the miR-155-deficient cells (Fig. 4F-H).This all points to the rapid changes in the cellular metabolism of MEC-1 cells under hypoxia, significantly depending on the presence of miR-155, whose deficiency accelerates the lactate metabolism elicited by hypoxia. ", "section_name": "Cellular metabolism of leukemic cells during hypoxia significantly depends on the presence of miR-155", "section_num": null }, { "section_content": "Hypoxia is a hallmark of cancer and is a hematopoietic niche that maintains proliferation of cells.Hypoxia modulates cell cycle, transcriptome and cellular metabolism.The metabolic response to hypoxia results in a shift of ATP production to glycolysis and lactate metabolism at the expense of oxidative phosphorylation [28].In this study, we aimed to describe how oncogenic miR-155 influences CLL cells in hypoxia in terms of gene expression profiling (mRNA/miRNA expression level).In general, there is not a uniform and exact opinion among researchers on the oxygen level (percentage) in the leukemic niche and its application for in vitro conditions in laboratory.From literature, it is known that the oxygen level in normal tissues varies between 1 and 11% and in tumors is about 2% [29].The B cells are characterized by intensive migration and circulation in the human body, from blood stream with almost normoxia into tissues (spleen, lymph nodes) with different hypoxia levels [30].In line with this, Koczula et al., 2016 performed a detailed analysis of oxygen levels in CLL cells in vivo by nuclear magnetic resonance (NMR) technique [4].They described that CLL cells are very flexible in different oxygen levels and could rapidly adapt by modulating their transcriptome and metabolome [4].The main message of this work with primary CLL cells points to very heterogeneous oxygen levels in the human body and extreme plasticity of CLL cells.CLL cells migrate (blood stream, lymph nodes, spleen, bone marrow) from almost normoxia in the circulating blood into lymph nodes where oxygen is about 3%.In order to determine the best range of oxygen levels that will be used in further experiments, we optimized the oxygen level (0.2%, 1% and 5%) and time periods (24 h-120 h) (Fig. 1A,B).Based on the highest expression level of hypoxia-related genes as LDHA, HK1, GLUT1, GLUT3 and the lowest expression of pro-apoptotic gene TP53INP1 we found the optimal in vitro hypoxic condition is at 1% oxygen and in the period of 24 h and 48 h (Fig. 1C).Similarly, Koczula et al., 2016 confirmed hypoxia by overexpression of mRNAs of LDHA, VEGF and GLUT1 in primary CLL cells [4].In the blood stream, the CLL cells are used to hypoxia and are characterized by high HIF-1α expression when entering into lymphoid tissues [4].In lymphoid tissues, the CLL cells are constantly supplied by essential signals for their survival and proliferation through interaction with accessory cells, usually stromal cells, contributing to drug resistance and apoptosis [31].The HIF-1α expression is induced also by PI3K and ERK mitogen-activated protein kinase (MAPK) signaling in stromal cells [32].Surprisingly, we were not able to detect increased level of HIF1α at mRNA (Fig. 1C) and at the protein level (Supplemental Fig. 2).We assume that it could be due to the increased mRNA level of miR-155 (in MEC-1 control cells in hypoxia), EGLN1, PIK3CA and low level of VHL as these are the key regulators of HIF1α and direct targets of miR-155 [23] or by another miRNA impairment.Another possible reason could be fast degradation of HIF1α during the first 24 h of hypoxia [39].In addition to transcription factors and other molecules, hypoxia modulates microRNAs, which react by their extreme expression.Among the well-described hypoxamiRNAs belongs miR-210 [17].MEC-1 cells react to hypoxia with a significantly elevated level of miR-210 (Figs.1C and3A) that is in concordance with other observations [17] and reviews [33].Another explanation for why we were not able to detect HIF1α could be its replacement by HIF-2 signaling after longer exposure of cells to hypoxia [17].Hypoxic conditions could be induced experimentally by using different methods and molecules [34].To validate the in vitro hypoxia accomplished in the hypoxic glove box we induced hypoxia in CLL (MEC-1) cells chemically.Thus, MEC-1 cells were treated with 2-OG analogue, dimethyloxalylglycine (DMOG), a competitive inhibitor of prolyl hydroxylase domain-containing proteins and with deferoxamine mesylate salt (DFO), an iron-chelating agent, both for 3 h, 6 h, and 24 h (Fig. 3A).Both chemicals significantly induced hypoxia in vitro in similar actions.Surprisingly, chemically-induced hypoxia resulted in more pronounced expression of hypoxiarelated genes as EGLN1, VEGFA, HK2 and GLUT3 (Fig. 3A) with moderate level of apoptosis (Fig. 3B) in comparison to the data from hypoxic glove box (Fig. 1C).Increased level of apoptosis by chemical inductors of hypoxia is in accordance with others [35].Increased cell proliferation in hypoxia, especially in cancer cells, is a common hallmark.Under normal conditions, cell proliferation decreases with low oxygen levels or stress.In addition, overexpression of HIF-1α results in cell cycle arrest in normal cells (lymphocytes, keratinocytes, embryonic stem cells, and hematopoietic stem cells) [36].However, the opposite situation happens in the malignant cells.In general, malignant cells proliferate at a high rate and express high levels of HIF-1α not only due to hypoxia but also due to deregulated signaling pathways that increase their survival [16].Here we detected significantly higher proliferation rates of MEC-1 cells in hypoxia depending on the presence of miR-155 (Fig. 2A,B) and accompanied by slightly increased apoptosis in the absence of miR-155 (Fig. 2C).One of the potential explanations could be deregulation of HIF-dependent miRNA regulatory network.Overexpression of HIF-1α induces the expression of oncomiRNAs in malignant cells [16].Sawai et al., 2022 selected 17 hypoxia related miRNAs (hypoxamiRNAs) in solid tumors, among them also miR-155 and miR-210.Similarly, these miRNAs were significantly changed in MEC-1 cells (Fig. 1C and Fig. 3A), especially miR-210, which was highly upregulated.MiR-155 represents the key oncomiRNA and has several combined roles in preventing apoptosis, modulating gene expression, blocking the phosphorylation of glucose, promoting many cancers and leukemias [19].There is evidence that miR-155 is actively involved in glucose metabolism in breast cancer, such that deletion of miR-155 abolishes glucose uptake [13].However, according to our data, MEC-1 cells reacted to hypoxia by increasing glucose uptake (Fig. 4A,B).This discordance could be due to different cell origin, breast cancer versus CLL where miR-155 could work in a different manner.Furthermore, the direct target of miR-155 is HK2 (hexokinase 2; by current MiRTarBase version 9.0, 2022; [37]) which phosphorylates glucose, thereby committing it to the glycolytic pathway.This is additional evidence that glucose metabolism is controlled by miR-155.We detected significantly increased levels of HK2 mRNA in miR-155 deficient MEC-1 cells (Fig. 1A,C).The absence of miR-155 results in increased glucose uptake and lactate production in MEC-1 cells in hypoxia (Fig. 4A,B,C).This notion is in accordance with experiments performed on the primary CLL cells, where the production of lactate was correlated with the glucose consumption [4].At the same time the mitochondrial oxidative phosphorylation was turned off in hypoxia and this effect was exacerbated by the deficiency of miR-155. To conclude, CLL cells are highly flexible and can adapt to different oxygen levels by coordinated changes at the mRNA/miRNA expression level and metabolism.One possible mechanism of this adaptation could be through the upregulation of glucose transporters (e.g., GLUT1 and GLUT3) that promotes glucose uptake and anaerobic metabolism and simultaneously shutsdown the mitochondrial energetic metabolism.These abovementioned effects are strengthened in the absence of miR-155. ", "section_name": "Discussion", "section_num": null }, { "section_content": "In the present study, we aimed to answer questions regarding the role of miR-155 (in terms of proliferation, metabolism and gene expression profile) in MEC-1 cells under hypoxic conditions.We confirmed the coupled effect of hypoxia and absence of miR-155 on the proliferation rate of MEC-1 cells, where the deletion of miR-155 significantly inhibits cell growth in normoxia and in hypoxia.Hypoxia-related genes such as EGLN1, VEGFA, HK2, HK1, LDHA and especially GLUT1, GLUT3 were upregulated in hypoxia.We detected the extreme upregulation of hypoxamiR-210 in MEC-1 cells under hypoxia.We showed the effect of miR-155 presence/ absence on glucose and lactate metabolism by qRT-PCR and by metabolic assays.Interestingly, the absence of miR-155 in MEC-1 cells stimulate the expression of mRNA of EGLN1, HK2, GLUT1 in the hypoxic glove box and GLUT3 in the chemically-induced hypoxia, which could be one of the possible adaptation mechanisms at different levels of hypoxia. ", "section_name": "Conclusions", "section_num": null } ]	[ { "section_content": "Authors thank to the research group led by Prof. Maria Hubalek Kalbacova (Laboratory of Interaction of Cells with Nanomaterials, The Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague) for their help with measurement of 2-deoxy-Glucose (2-NBDG).Further authors thank students of medicine Ms. Virginia Cox and Mr. Cory Eaton, two native English speakers from the First Faculty of Medicine, Charles University in Prague for their excellent help with English language of the manuscript.Authors also thank to Prof. Martin Vokurka (Head of The Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague) for the institutional support. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "This research was supported by the Charles University, Czech Republic grants PRIMUS/17/MED/16 (concretely researchers E.G. and K.S.V) and START/ MED/089 (concretely researchers E.G., L.D., T.H. and K.S.V), and by Youth and Sports grant of the Czech Republic PROGRES Q26/LF1 (concretely researchers E.G., L.D., T.H, M.B. and K.S.V).Next by AIRC (Italian Association for Cancer Research), Grant: IG21408 (concretely R.J. and C.R.). ", "section_name": "Funding", "section_num": null }, { "section_content": "All data generated or analyzed during this study are included in this published article [and its supplementary information files]. ", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s12935-024-03437-8.Supplementary Material 1. Figure S1: Description of creation of miR-155 deficient MEC-1 cells and its validation.Supplementary Material 2. Figure S2: Immunoblot.Supplementary Material 3. Figure S3: Representative dot plot pictures to all flow cytometry data within manuscript.Supplementary Material 4. Tables. Conceptualization, K.S.V.; methodology, E.G., L.D., T.H., M.B., V.G., R.J., S.F.; formal analysis, E.G., T.H., L.D, M.B.V.G., R.J., S.F.; investigation, E.G., L.D., T.H., M.B., V.G., R.J., S.F.; writing-original draft preparation, E.G., K.S.V.; writing-review and editing, E.G., L.D., T.H., C.R., M.C.; visualization, E.G., L.D., T.H.; supervision, K.S.V.; project administration, E. G, K.S.V.; funding acquisition, K.S.V., E.G., C.R. All authors have read and agreed to the published version of the manuscript. Ethics approval and consent to participate Not applicable. The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Supplementary Information", "section_num": null }, { "section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s12935-024-03437-8.Supplementary Material 1. Figure S1: Description of creation of miR-155 deficient MEC-1 cells and its validation.Supplementary Material 2. Figure S2: Immunoblot.Supplementary Material 3. Figure S3: Representative dot plot pictures to all flow cytometry data within manuscript.Supplementary Material 4. Tables. ", "section_name": "Supplementary Information", "section_num": null }, { "section_content": "Conceptualization, K.S.V.; methodology, E.G., L.D., T.H., M.B., V.G., R.J., S.F.; formal analysis, E.G., T.H., L.D, M.B.V.G., R.J., S.F.; investigation, E.G., L.D., T.H., M.B., V.G., R.J., S.F.; writing-original draft preparation, E.G., K.S.V.; writing-review and editing, E.G., L.D., T.H., C.R., M.C.; visualization, E.G., L.D., T.H.; supervision, K.S.V.; project administration, E. G, K.S.V.; funding acquisition, K.S.V., E.G., C.R. All authors have read and agreed to the published version of the manuscript. ", "section_name": "Author contributions", "section_num": null }, { "section_content": "Ethics approval and consent to participate Not applicable. ", "section_name": "Declarations", "section_num": null }, { "section_content": "", "section_name": "Consent for publication", "section_num": null }, { "section_content": "", "section_name": "Not applicable", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.1038/s41598-019-41805-x	Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice	<jats:title>Abstract</jats:title><jats:p>Insertional mutagenesis is a powerful means of identifying cancer drivers in animal models. We used the Sleeping Beauty (SB) transposon/transposase system to identify activated oncogenes in hematologic cancers in wild-type mice and mice that express a stabilized cyclin E protein (termed cyclin ET74AT393A). Cyclin E governs cell division and is misregulated in human cancers. Cyclin ET74AT393A mice develop ineffective erythropoiesis that resembles early-stage human myelodysplastic syndrome, and we sought to identify oncogenes that might cooperate with cyclin E hyperactivity in leukemogenesis. SB activation in hematopoietic precursors caused T-cell leukemia/lymphomas (T-ALL) and pure red blood cell erythroleukemias (EL). Analysis of >12,000 SB integration sites revealed markedly different oncogene activations in EL and T-ALL: <jats:italic>Notch1</jats:italic> and <jats:italic>Ikaros</jats:italic> were most common in T-ALL, whereas ETS transcription factors (<jats:italic>Erg</jats:italic> and <jats:italic>Ets1</jats:italic>) were targeted in most ELs. Cyclin E status did not impact leukemogenesis or oncogene activations. Whereas most SB insertions were lost during culture of EL cell lines, <jats:italic>Erg</jats:italic> insertions were retained, indicating Erg’s key role in these neoplasms. Surprisingly, cyclin ET74AT393A conferred growth factor independence and altered Erg-dependent differentiation in EL cell lines. These studies provide new molecular insights into erythroid leukemia and suggest potential therapeutic targets for human leukemia.</jats:p>	[ { "section_content": "tissue architecture and suppressed tumorigenesis.Cyclin ET74AT393A expression also caused ineffective erythropoiesis with marked expansion of immature erythroid precursors in the spleen and bone marrow, impaired erythroid differentiation, and mild anemia.These features resemble the early stages of human refractory anemia/ myelodysplastic syndrome (MDS). Because MDS can evolve to leukemia in humans, we speculated that cyclin ET74AT393A mice may provide a sensitized background to identify genetic events that cooperate with abnormal cyclin E regulation to promote leukemia.We thus used interferon-inducible Mx-Cre to activate the SB transposase in hematopoietic precursors to identify genes that might cooperate with abnormal cyclin E regulation to promote leukemia.The stabilized cyclin E allele neither predisposed mice to hematologic cancers nor altered gene activations by SB.Strikingly however, Mx-Cre-induced SB activation caused highly penetrant hematologic cancers within 8-13 weeks after Cre induction.To control for biases in transposon integrations that frequently occur proximal to the T2Onc array 15,16 , we used two different T2/Onc2 strains that contained the transposon array on different chromosomes.The most common malignancies were immature T-cell leukemia/lymphomas (T-ALL) and pure red blood cell erythroleukemias (EL), and there was a non-significant trend towards more EL in the cyclin ET74AT393A mice. To identify activated oncogenes in these neoplasms, we determined the transposon insertion sites in all ELs and T-ALLs.Transposon insertions that are shared by multiple independent tumors, termed common insertion sites (CIS), often occur in the vicinity of cancer-associated genes, which provides the selective pressure for these shared insertions.We identified CIS using two different statistical methods and found that the CIS profile of ELs and T-ALLs differed markedly.Whereas Notch and Ikaros insertions were most common in T-ALL, ETS family transcription factors (Erg and Ets1) were the most commonly activated genes in ELs and were activated in the almost all of these tumors. While T-ALL is common in SB screens performed in blood cells, EL has not, and we thus examined EL in more detail by developing 5 transplantable EL cell lines.Transposon analyses indicated that the vast majority of CISs found in primary ELs were lost during culture of EL cell lines, suggesting that they were not required for their proliferation and maintenance in vitro.However, all EL lines retained their Erg insertions and overexpressed ERG protein, further supporting the key role of Erg activation in EL.Insertions near the Bach2 transcription factor were also retained in several cell lines, and one EL line also retained a Flt3 insertion and exhibited FLT3-dependence.Finally, although cyclin ET74AT393A expression did not impact leukemogenesis or CIS involvement, we found two phenotypic differences between EL cell lines derived from WT and cyclin ET74AT393A mice.First, while WT EL lines remained dependent on exogenous growth factors (GFs), the cyclin E mutant cell lines did not and proliferated in the absence of GFs.Next, Erg knockdown resulted in erythroid differentiation of the WT but not the cyclin ET74AT380A cell lines.These findings suggest that properly regulated cyclin E activity influences both growth factor requirements and differentiation in EL cells. ", "section_name": "", "section_num": "" }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "We performed a SB insertional mutagenesis screen in WT and cyclin ET74AT393A mice.Mice homozygous for the cyclin ET74AT393A mutation and Rosa26SB11 LSL SB transposase and containing one of two different T2/Onc2 arrays were crossed with mice homozygous for cyclin ET74AT393A and expressing the Mx1-Cre transgene, which generated the experimental cohort (cyclin ET74AT393A; Rosa26SBLSL; T2/Onc2+; Mx1-Cre1) (Fig. 1a).The matched control cohort was wild-type cyclin E; Rosa26SBLSL; T2/Onc2+; Mx1-Cre1.Parallel crosses were made using two separate T2/Onc2 lines with transposon arrays on different chromosomes (strain 6113-chromosome 1, strain 6070 -chromosome 4) 17,18 Mice (7-10 weeks old) were injected with poly I:C to activate Cre recombinase expression and subsequent transposase expression.Mx1-Cre induction and SB expression was monitored by loss of GFP expression in peripheral blood leukocytes from a subset of study mice, which demonstrated 71-86% loss of GFP-expressing cells following induction (Table S1). SB insertional mutagenesis in immature hematopoietic cells resulted in progressive serious illness, including weight loss, poor grooming, and hunched posture, leading to euthanasia within 3 months of Mx-Cre induction.Necropsy showed hematologic malignancies in all animals, including lymphoid, myeloid, erythroid and megakaryocytic tumors (Fig. 1c, Table S2).Surprisingly, rather than accelerating tumorigenesis, the cyclin ET74AT393A allele imparted a non-significant trend towards longer survival (11.6 weeks post injection for wild-type and 12.9 weeks for cyclin ET74T393A mice, p = 0.08) (Fig. 1b).The most frequent neoplasm was immature T-cell lymphoblastic lymphoma with leukemia (T-ALL), which usually presented with a massively enlarged thymus.T-ALL typically involved the spleen and bone marrow and in some instances, disseminated to the liver, kidneys and lungs.Leukemia was also noted with leukocytosis and circulating lymphoblasts.Histologic sections of affected organs revealed homogeneous tumors composed of tightly packed lymphoblasts with a high mitotic rate and scattered apoptotic cells admixed with phagocytic histiocytes (Fig. S1).Immunophenotyping by flow cytometry showed expression of CD3 and CD8 with frequent variable co-expression of CD4, findings consistent with immature T-cell lymphoblastic leukemia (Fig. 2f,g, Fig. S1, Table S2). Many (40%, n = 100) mice developed EL with intermixed megakaryocytic differentiation, which caused prominent splenomegaly and involved the bone marrow and sometimes the liver (Figs 1c, 2a,b,e and Table S2).The mice with EL exhibited peripheral involvement with leukocytosis consisting of a prominent population of circulating erythroblasts with dark basophilic cytoplasm (Fig. 2c,d).Multiparameter flow cytometry studies revealed immature erythroblasts (as shown by CD45/side scatter) with dim CD45 expression, variable CD117 (c-Kit) expression, bright CD71 (transferrin receptor) expression, and occasional Ter119 (glycophorin associated protein) expression (Fig. 2f,g, Table S2).ELs varied somewhat with respect to their degree of erythroid differentiation, with variable expression of CD117 (immature immunophenotype) and Ter119 (mature immunophenotype) (Fig. S2).The immunophenotype of the neoplastic population was identical in samples isolated from peripheral blood, spleen and bone marrow (Fig. S3).Some ELs showed megakaryocytic differentiation with scattered enlarged cells with abundant cytoplasm and multilobulated nuclei, morphologically consistent with megakaryocytes (Fig. 2e).Additional flow cytometry studies showed variable expression of CD41, consistent with megakaryocytic differentiation in these cases (Fig. S4).Overall, these tumors displayed many features resembling those of pure red blood cell EL in humans (see Discussion) 19 .Some mice (7%) contained mixed tumors of both lymphoid and erythroid lineages identified by parallel flow cytometry studies (Table S2).The spectrum of hematologic malignancies was similar in the cyclin E-T74AT393A and wild-type mice, with a small trend toward increased EL in the mutant cyclin E mice (Fig. 1c). ", "section_name": "Development and Characterization of SB-induced hematologic cancers.", "section_num": null }, { "section_content": "To identify CIS, we isolated DNA from 40 ELs (spleens) and 53 T-ALLs (thymus or lymph node), amplified the transposon integration sites via ligation-mediated PCR, and identified the transposon integration sites by next generation sequencing.We mapped 5002 EL insertion sites and 7012 T-ALL sites (Table S3).Each tumor contained between 50 and 150 unique insertions, presumably representing both driver and passenger insertions.We identified CIS by using two different statistical methods that differ in the parameters used to identify CIS and their associated genes: (1) gene-centric common insertion site analysis (gCIS), and (2) TAPDANCE 20,21 . gCIS identified 74 total CIS in T-ALLs (Table S4).The chromosome that contains the transposon concatemer (the \"donor chromosome\") is disproportionately targeted by transposon insertions (\"local hopping\") in SB screens.As expected, many CIS were mapped to the donor chromosome.Although these insertions could represent true genetic drivers of cancer, we initially filtered these CIS from subsequent analyses, resulting in 25 T-ALL CIS (Table 1).This analysis identified many established drivers of T-ALL, including Notch1, Ikzf1, Rasgrp1, and Akt2 6 .The presence of the cyclin E mutation did not impact the spectrum of CIS observed.Since T-ALLs have been well studied in insertional mutagenesis studies, we focused on the ELs for the remainder of our study 4,[22][23][24] . gCIS identified 36 CIS genes in ELs prior to filtering out the donor chromosome CIS (Table S5), and 13 CIS after donor chromosome filtering (Table 2).Many of the EL CIS thus fell within the donor chromosomes.We observed a very high insertion rate in the ETS family of transcription factors (TFs), with insertions in Erg and Ets1 in 65% and 44% of ELs, respectively.In fact, only 4 of the 40 ELs lacked insertions targeting either Erg or Ets1 (Table S4).Interestingly, a previous SB insertional mutagenesis study performed in Jak2 mutant mice also identified ETS transcription factors as CIS in EL, and Erg has been implicated in human EL (see discussion), highlighting the importance of ETS TFs in EL 25 .Many CIS occurred near genes implicated in hematologic cancers and hematopoietic development (Erg, Ets1, Epo, Il2rb, Flt3, Kras, Stat5b, Fli1).While CIS within donor chromosomes were filtered from our analyses, some of the CIS found within donor chromosomes have been reported in other transposon-mediated insertion studies targeting hematologic neoplasms, and thus may represent physiologically significant gene alterations rather than local hopping.Examples of these types of CIS (on chromosome 4) that are likely to have contributed to EL in our study include Csf3r (35% of ELs), Bach2 (41% of ELs) and Cdkn2a/Arf (5% of ELs).Because we found each of these CIS in both T2Onc strains, these insertions were not restricted to potential local hops.TAPDANCE, an alternate statistical method that eliminates donor chromosome insertions, was also used to annotate CIS and gene associations in T-ALL and EL.The most frequent CIS found by TAPDANCE in T-ALL included Notch1, Ikzf1, Rasgrp1, Akt2, Runx1, FoxP1, whereas the most frequent CIS present in EL were Erg and Ets1, followed by Epo, Gata1, Pik3ca and Fli1 (Tables S6 andS7). CIS evolution in EL cell lines.We used marrow transplantation to demonstrate that ELs were truly neoplastic and capable of self-replication.Five primary ELs were transplanted into sub-lethally irradiated mice, and recipient mice developed signs of progressive leukemia with a latency of 10-20 days post-injection.Analysis of peripheral blood revealed similar features to the parental tumors, including circulating erythroblasts with the same immunophenotypes as the original tumor, albeit more homogeneously (Fig. 3a).Necropsy of the transplanted mice demonstrated splenomegaly, similar to that seen in the original model.Stable EL cultures were established from five of these transplanted mice using murine erythroblast cell culture media containing c-Kit ligand and erythropoietin.No major immunophenotypic or morphologic changes occurred during the prolonged cell culture (Fig. 3a, Table S2).We used these long-term EL cultures to further identify putative driver insertions that promote EL.Since the SB transposase is constitutively active in these cell lines, we predicted that serial passage might result in the loss of irrelevant or passenger transposon insertions that were not required for EL proliferation or maintenance in culture, and conversely, that insertions required for tumor maintenance would be preserved.All five EL primary cultures were cultured for 12 weeks in the presence of growth factors and then analyzed for transposon insertions as described above (Table S3).Long-term cell culture dramatically altered the number and identity of the insertions.On average, 113 unique insertions (ranging between 57-143) were identified from the original ELs.Following long-term culture, the number of insertions decreased to an average of 24 unique insertions (ranging from 13-39) per cell line, and only a few of these (1-4) had been previously identified as CIS (Fig. 3b,c).The loss of insertions may result from continued transposon hopping and tumor evolution in culture, or may alternatively reflect clonal selection from an initially heterogeneous population of cells in primary ELs.In all cases, just a few previously identified CIS were maintained during long-term culture (Fig. 3b,c and Table S8).In one cell line, we detected an Erg insertion that had not been found in the primary tumor, which may reflect either a small subclonal population present in the original tumor that was not detected in the initial sequencing, or alternatively, clonal evolution in vitro.Of the previously identified CIS, only Erg insertions were retained in all the cell lines tested, suggesting that persistent Erg deregulation is required for EL proliferation and/or maintenance in culture.Interestingly, Bach2, a chromosome 4 insertion site that was found in both T2Onc strains, was identified as a CIS in 3/5 cell lines, suggesting that this hematopoietic TF may have an important role in EL 24,26 . ", "section_name": "Common Insertion Site (CIS) Identification in T-ALL and EL.", "section_num": null }, { "section_content": "Erg was the most common EL CIS in our study, with 65% of tumors containing at least one Erg insertion.These integrations were found in the same transcriptional orientation as the ERG gene and upstream of exons 3 and 4, suggesting that the insertions led to overexpression of truncated Erg mRNAs and proteins (Fig. 4a). Erg overexpression drives murine erythroid-megakaryocytic leukemia and has been associated with various human leukemias and poor outcomes [27][28][29] .We used shRNA to reduce ERG protein expression to study the role of persistent Erg deregulation in long-term EL cultures.Primary EL cell lines expressed variable levels of ERG protein, and Erg shRNA only partially reduced ERG abundance (Fig. 4b).The inability to fully silence Erg expression may reflect the essential role of Erg deregulation in these cells.Partial Erg knockdown induced immunophenotypic differentiation in the cell lines derived from the WT background, with decreased CD117 (cKit) expression and increased Ter119 (glycophorin B), consistent with erythroid maturation (Fig. 4b,c).These results are consistent with prior studies that have shown that ERG expression in human and murine leukemias inhibits differentiation [30][31][32] .Surprisingly, unlike WT EL cell lines, EL lines derived from cyclin ET74AT393A mice did not exhibit immunophenotypic markers of differentiation after partial ERG knockdown (Fig. 4c).Even more unexpectedly, both cell lines derived from the cyclin E mutant mice proliferated without growth factor supplementation, while the three cell lines derived from wild-type mice required erythropoietin and c-Kit supplementation (Fig. 4d).These results suggest that inappropriate cyclin E regulation confers growth factor independence.This is reminiscent of observations with the related cell cycle protein, cyclin D2, which promotes growth factor independence in B-cell lines 33 .Thus, despite the similar disease spectrum in both strains, cyclin ET74AT393A expression overcame normal growth factor dependency, indicating that cyclin E may be a key mediator of mitogenic signaling in EL cells.Flt3 (FMS-like tyrosine kinase 3) is a receptor tyrosine kinase that is mutated in AML and confers a poor clinical prognosis 34 .We identified three Flt3 insertions in ELs, all of which were located downstream of exon 9 (Fig. 5a).These insertions are predicted to result in overexpression of a truncated FLT3 protein containing only the tyrosine kinase domain without the receptor domain.One of the tumors selected for culture (4489) had the Flt3 insertion and retained this insertion after serial passage.Western blot analysis shows that this cell line over-expressed a truncated ~65 kDa form of FLT3 protein without apparent expression of endogenous full length Flt3 (Fig. 5b).To test if the Flt3 insertion is required for proliferation of this EL line, we treated all five of the tumor-derived cell lines with three different pharmacologic inhibitors that target the FLT3 kinase and have been used in clinical trials of patients with FLT3 mutant AML (Lestaurtinib, PKC412, and Sorafenib).Compared with the other cell lines, line 4489 (with the Flt3 insertion) was exquisitely sensitive to all three inhibitors, showing that this EL cell line remained dependent on FLT3 activity for proliferation (Fig. 5c-e). ", "section_name": "Effects of Erg insertions and cyclin ET74AT393A expression on EL differentiation and growth factor requirements.", "section_num": null }, { "section_content": "Based on our previous work showing that mice with a stabilized cyclin E protein develop proliferative and differentiation anomalies in hematopoietic cells, we undertook this study to use SB to identify putative cyclin E-cooperating oncogenes.In light of the phenotypic consequences of cyclin ET74AT393A expression in mice, as well as the roles for cyclin E in human cancer and murine cancer models 7,14,[35][36][37] , it was surprising to find that the cyclin ET74AT393A allele did not alter the incidence, latency, or spectrum of hematologic cancers, or the gene activations observed.The reasons why cyclin ET74AT393A failed to contribute to multistep tumorigenesis in this model remain unclear.The T74AT393A mutation only stabilizes cyclin E in catalytically active complexes, and because many cyclin E-CDK2 complexes are inactive, this mutation has a greater impact on cyclin E-CDK2 activity than cyclin E abundance.In contrast, cyclin E amplifications in human tumors elevate cyclin E abundance, which may or may not increase cyclin E activity (due to compensatory mechanisms that suppress CDK2 activity). Because cyclin E has non-catalytic activities (e.g.replication origin licensing), one possibility is that mutations that differentially impact cyclin E abundance versus activity may lead to different biologic outcomes 38 .However, we do not have evidence to directly support this speculation from this study. While cyclin ET74AT393A expression did not alter carcinogenesis, it did impact growth factor requirements and differentiation of EL cell lines.Cyclin E-CDK2 activation has previously been proposed to be a crucial feature of the G1 restriction point, which is defined at the point in the G1 phase of the cell cycle after which cells no longer require extracellular mitogens for S-phase entry 39,40 .Our findings that cyclin ET74AT393A rendered two EL cell lines growth factor independent supports this notion, although these studies involved a limited number of cell lines.Of note, our attempts to convert WT-EL cell lines to GF independence through ectopic cyclin ET74AT393A expression were unsuccessful because ectopic cyclin ET74AT393A expression was toxic.SB activation by Mx-Cre caused two distinct hematologic neoplasms, T-ALL and EL, that differed morphologically, immunophenotypically, and with respect to the identified CIS associated with each disease.While T-ALL has been extensively described in SB models 22 , EL is much less common 25 .In humans, acute erythroleukemia (EL) is a rare and relatively poorly characterized form of acute myeloid leukemia 19 .Until recently, the diagnostic criteria for EL encompassed a diverse set of hematopoietic malignancies that share some degree of erythroid hyperplasia, ranging from an expanded abnormal myeloid blast population with erythroid hyperplasia to a pure erythroid leukemia with erythroid blasts encompassing greater than 80% of the marrow cellularity (2008 WHO; see below).The prior diagnostic criteria for acute erythroleukemia thus included both erythroleukemia (erythroid/myeloid) and pure erythroid leukemia, which occurs less commonly.Because erythroleukemia (erythroid/ myeloid) frequently arises from pre-existing MDS (and shares many clinical features of MDS), the WHO recently modified the diagnostic criteria for EL: the more common erythroleukemia (erythroid/myeloid) is now classified as MDS or AML based on the percentage of myeloid blast population, irrespective of the erythroid component 41,42 .Therefore, many cases that had been previously diagnosed as EL are now reclassified as low grade MDS.Pure erythroid leukemia (PEL) remains a distinct diagnostic entity per WHO criteria and is characterized by malignant immature erythroid cells that represent greater than 80% of the marrow.Because of its rare occurrence and previously imprecise diagnostic criteria, our understanding of PEL remains limited.A few small-scale clinical studies on PEL have demonstrated that the leukemic cells usually have complex chromosomal abnormalities, frequent p53 mutations, and extremely poor overall survival 43,44 . The murine EL that developed in this study most resembled human PEL, with an expanded erythroid blast population (>80% of the marrow cellularity) associated with peripheral cytopenia, circulating erythroid blasts and frequent extramedullary involvement of the liver and spleen.We identified many insertion sites in ELs that target genes involved in myeloproliferative disorders, growth factor signaling, and signal transduction.In accordance with previous work on murine and human EL [27][28][29] , our studies support a crucial role for ETS transcription factors in the development and maintenance of EL.Most of the ELs that we studied (90%) had inserts in one of the ETS transcription factors (ERG, ETS, or FLI1) and it is tempting to speculate that the few ELs without these insertions may have activated these pathways through other mechanisms.However, we do not have direct evidence to support this speculation.Strikingly, ERG-associated SB insertions were retained in each of the five EL cell lines that we derived from primary ELs, while the vast majority of other CIS found in the primary tumors were not (Fig. 3c).This supports the idea that EL cell lines depend upon continued ERG deregulation, which is consistent with the differentiation caused by partial ERG knockdown (Fig. 4c).Our studies also suggest that Erg may be a good therapeutic target for several forms of AML, including PEL.The other CIS that was retained in multiple EL cell lines was Bach2 (B-lymphoid transcription factor, BTB and CNC Homology 1 Basic Leucine Zipper Transcription Factor 2), a transcriptional repressor that regulates antibody class switching and has been implicated in B-cell neoplasm 45,46 .Bach1 and Bach2 are highly regulated and homologous proteins with key functions in iron hemostasis, erythropoiesis, megakaryopoiesis, and autoimmunity 47 .Our finding that Bach2 insertions were retained in EL cell lines suggests a biologic selection for persistent Bach2 deregulation and perhaps a role for Bach2 in EL development and/or maintenance.During erythropoiesis, elevated levels of free heme bind to and inhibit Bach1, thereby activating the expression of Beta-globin and other genes involved in erythroid differentiation 48 .Although Bach2 functions as a transcriptional repressor during B-cell maturation and has been implicated in B-cell neoplasms, other studies have shown the Bach2 is also regulated by heme binding and that overexpression of Bach2 in hematopoietic stem cells promotes erythroid commitment, suggesting it may also play an early role in directing erythroid lineage fate decisions 49 . Finally, pure erythroid leukemia is an aggressive poorly characterized form of AML.Now that the WHO has redefined PEL as a distinct form of AML, new clinical studies are needed to identify the molecular pathways that promote PEL.The functional studies presented here provide candidate genes and pathways that should serve as a resource for future studies of human PEL and help define altered pathways that promote this rare, but highly aggressive, leukemia. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Mice.All mice used in these studies were previously described.Homozygous cyclin ET74AT393A mice were developed and maintained as described 13 .The Cre inducible RosaSBase-LSL transposase and the T2/Onc2 transposon (Lines 6113 and 6070) mice were obtained from N. Copeland 17,18 .Mx1-Cre transgenic mice were obtained from Jackson Laboratories (Bar Harbor, ME).The breeding schemes to generate study mice are shown in Fig. 1a.For Mx1-Cre induction, mice received intraperitoneal injections of polyinosinic: polycytidylic acid (pIpC) (10 μg/g body weight) every other day for a total of five injections.For transplant studies, erythroleukemia cells were expanded in vitro prior to injection.Recipient C57/Bl6 mice were sublethally irradiated (700 Gy) 24 hours prior to transplantation and infused with 1 × 10 6 cultured erythroleukemia cells.Each cell line was transplanted into five recipient mice.Mice were monitored daily using institutional standard procedures and euthanized when indicated.Animal studies and all animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and were carried out at the Fred Hutchinson Cancer Research Center (FHCRC).All methods were performed in accordance with the guidelines and regulation established by FHCRC IACUC, Institutional Review, and Institutional Biosafety Committees. Histopathological and Hematological Analysis.Mice were euthanized via carbon dioxide inhalation and necropsies were performed.All tissues were harvested and fixed in 10% formalin for 5-7 days and stored in 70% ethanol until submitted for pathological evaluation.Sternums for bone marrow analysis were decalcified in Formical-4 (Decal Chemical Corp) for 24 hrs.after fixation in formalin.After fixation, all samples were submitted to the Experimental Histopathology Core Facility at FHCRC where tissues were processed, embedded in paraffin, cut into slides, and stained with H&E according to standard techniques.Tissues were reviewed by a board-certified hematopathologist (KRL) and a board-certified veterinary pathologist (SEK).Peripheral blood was harvested and analyzed via Siemens Advia 2120i with multispecies software and a manual differential analysis.Lymphomas and leukemias were classified using the Bethesda recommendations for mice 50,51 . Flow cytometry.Primary splenocytes and thymocytes were recovered by crushing excised spleen or thymic tissue between glass microscope slides, washing with PBS + 2% FBS, and passing through an 80 µm mesh filter (Sefar, Nitex 03-80/37).Cells were washed once in PBS + 2% FBS and were resuspended in PBS + 2% FBS and kept on ice until analysis.For analysis of bone marrow, marrow was extracted from femurs using a mortar and pestle and processed as above.For analysis of peripheral blood, red blood cells were lysed (155 mM NH 4 CL, 12 mM NaHCO 3 , 0.13 mM EDTA) at room temperature and washed twice in PBS + 2% FBS.Flow cytometry analysis included a 4-color T-cell panel (CD8a-PE, CD4-APC, CD19-APC-Cy7and CD45r/B220-PerCP (BD Biosciences)) and a 6-color erythroid/myeloid panel (CD45-APC-Cy7, CD71-FITC, CD117-PE-Cy7, Ter119-APC, Ly-6G/Ly-6C (Gr1)-PerCP-Cy5.5, and B220-FITC (BD Biosciences) and CD3e Alexa Fluor 488 (BioLegend).Cells were also stained with DAPI to determine cell viability.Flow cytometry was performed on a Canto 2 (Becton Dickinson) or a custom designed LSR2 (Becton Dickinsin, Franklin Lakes, NJ) and analyzed using either FlowJo Software (FlowJo, Ashland, OR) or Woodlist, a noncommercial software program developed in our clinical laboratory 52 . Ligation-mediated PCR to amplify transposon junction sequences.Genomic DNA was extracted from frozen tissue using the Puregene Core Kit A (Qiagen) according to manufacturer's instructions.Five μg of genomic DNA in a volume of 100 μl of ddH2O was sheared to 300 bp lengths using the Covaris E-series sonicator according to the manufacturer's protocol.One μg of DNA (20 μl) was end-repaired in a reaction containing T4 DNA polymerase and polynucleotide kinase and incubated at 20°C for 30 minutes.Then 1 μl of 0.5 M EDTA was added and enzymes were heat-inactivated at 75°C for 20 minutes.Blunt/Alu adaptors were ligated to ends of repaired DNA and amplification of transposon junctions was conducted using a ligation-mediated PCR approach as previously described 20,22 .Individual sample library quality was analyzed by running an aliquot via agarose gel electrophoresis.Remaining PCR products were purified, pooled (20-25 ng per sample), and submitted for direct sequencing on an Illumina HiSeq 2000.Sequencing reads were analyzed using gene-centric common insertion site analysis (gCIS) or TAPDANCE 20,21 . ", "section_name": "Materials and Methods", "section_num": null } ]	[ { "section_content": "This work was supported by the following grants: NIH R01 CA193808 (B.E.C.), NIH 1R21CA229922 (A.J.D), and American Cancer Society 125006-RSG-13-183-01-CSM (J.E.G).This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA015704 and a pilot grant from Core Center of Excellence in Hematology P30 DK 56465.B.E.C is supported by the Rosput Reynolds Endowed Chair. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Cell lines.Primary tumor cell lines were derived by plating splenocytes in IMDM media containing 20% FBS, 4.5 × 10 -5 M monothioglycerol, 2 U/ml erythropoietin (Epoetin alpha; Amgen Biotechnology), and 50 ng/ ml mouse stem cell factor.Cell lines were then maintained in culture as indicated in the text.Lentivirus infection with Erg-specific shRNA, and retroviral infection with cyclin ET74AT393A and wildtype cyclin E were performed as previously described 53 . Western Blotting.Western blotting was performed as described 11 .Anti-ERG antibody (CM421AC) was from Biocare Medical (Pacheco, CA) and anti-FLT3 antibody (8F2) was from Cell Signaling (Danvers, MA). Cell lines were incubated with the indicated concentrations of drugs and incubated for 72 hours.Viability was measured using a resazurin-based assay and % viability was normalized to the untreated control. K.L. co-wrote the main manuscript, contributed to preparing all figures and tables, and performed experiments for each section of the manuscript.He also provided hematopathology interpretations for all flow and pathologic specimens.B.H. contributed to the main text and performed many of the mouse and sequencing experiments.B.F. assisted with the mouse colony, crosses, and genotyping.N.O.provided technical assistance for Figures 4 and5 and flow cytometry.S.K.-provided expert veterinary pathology services.J.G.-contributed to the development of the mouse strategies and crosses and edited the manuscript.L.D. assisted with DNA sequencing and analyses.A.S. provided statistical guidance for the analyses of insertion sites with TAPDANCE.A.D. provided expertise and oversight in all aspects of Sleeping Beauty strategy and analyses, assisted in analyses of transposon insertions through gCIS, coordinated sequencing strategies, and edited the manuscript.B.C. oversaw the entire project and co-wrote the manuscript and co-prepared all figures and tables. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-41805-x. The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Cytotoxicity Assays.", "section_num": null }, { "section_content": "Cell lines.Primary tumor cell lines were derived by plating splenocytes in IMDM media containing 20% FBS, 4.5 × 10 -5 M monothioglycerol, 2 U/ml erythropoietin (Epoetin alpha; Amgen Biotechnology), and 50 ng/ ml mouse stem cell factor.Cell lines were then maintained in culture as indicated in the text.Lentivirus infection with Erg-specific shRNA, and retroviral infection with cyclin ET74AT393A and wildtype cyclin E were performed as previously described 53 . Western Blotting.Western blotting was performed as described 11 .Anti-ERG antibody (CM421AC) was from Biocare Medical (Pacheco, CA) and anti-FLT3 antibody (8F2) was from Cell Signaling (Danvers, MA). ", "section_name": "", "section_num": "" }, { "section_content": "Cell lines were incubated with the indicated concentrations of drugs and incubated for 72 hours.Viability was measured using a resazurin-based assay and % viability was normalized to the untreated control. ", "section_name": "Cytotoxicity Assays.", "section_num": null }, { "section_content": "K.L. co-wrote the main manuscript, contributed to preparing all figures and tables, and performed experiments for each section of the manuscript.He also provided hematopathology interpretations for all flow and pathologic specimens.B.H. contributed to the main text and performed many of the mouse and sequencing experiments.B.F. assisted with the mouse colony, crosses, and genotyping.N.O.provided technical assistance for Figures 4 and5 and flow cytometry.S.K.-provided expert veterinary pathology services.J.G.-contributed to the development of the mouse strategies and crosses and edited the manuscript.L.D. assisted with DNA sequencing and analyses.A.S. provided statistical guidance for the analyses of insertion sites with TAPDANCE.A.D. provided expertise and oversight in all aspects of Sleeping Beauty strategy and analyses, assisted in analyses of transposon insertions through gCIS, coordinated sequencing strategies, and edited the manuscript.B.C. oversaw the entire project and co-wrote the manuscript and co-prepared all figures and tables. ", "section_name": "Author Contributions", "section_num": null }, { "section_content": "Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-41805-x. ", "section_name": "Additional Information", "section_num": null }, { "section_content": "The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Competing Interests:", "section_num": null } ]
10.1371/journal.pone.0112275	KCTD10 Is Involved in the Cardiovascular System and Notch Signaling during Early Embryonic Development	As a member of the polymerase delta-interacting protein 1 (PDIP1) gene family, potassium channel tetramerisation domain-containing 10 (KCTD10) interacts with proliferating cell nuclear antigen (PCNA) and polymerase δ, participates in DNA repair, DNA replication and cell-cycle control. In order to further investigate the physiological functions of KCTD10, we generated the KCTD10 knockout mice. The heterozygous KCTD10(+/-) mice were viable and fertile, while the homozygous KCTD10(-/-) mice showed delayed growth from E9.0, and died at approximately E10.5, which displayed severe defects in angiogenesis and heart development. Further study showed that VEGF induced the expression of KCTD10 in a time- and dose-dependent manner. Quantitative real-time PCR and western blotting results revealed that several key members in Notch signaling were up-regulated either in KCTD10-deficient embryos or in KCTD10-silenced HUVECs. Meanwhile, the endogenous immunoprecipitation (IP) analysis showed that KCTD10 interacted with Cullin3 and Notch1 simultaneously, by which mediating Notch1 proteolytic degradation. Our studies suggest that KCTD10 plays crucial roles in embryonic angiogenesis and heart development in mammalians by negatively regulating the Notch signaling pathway.	[ { "section_content": "KCTD10 is a member of the polymerase delta-interacting protein 1 (PDIP1) gene family [1], which consists of 3 members, PDIP1, KCTD10 and TNFAIP1 [2][3][4][5].All the three members contain a conserved BTB/POZ domain, a potassium channel tetramerisation (K-tetra) domain (a relative of BTB/POZ domain) at the N-terminus, and a proliferating cell nuclear antigen (PCNA)-binding motif at the C-terminus [1,4].KCTD10 is inducible by TNF-a, interacts with PCNA and the small subunit (p50) of DNA polymerase d [3].In A549 lung adenocarcinama cells, knockdown of KCTD10 decreases PCNA expression [6].Promoter analysis showed that KCTD10 can be regulated positively by SP1 and negatively by AP-2 transcription factors [5].In a recent study, KCTD10 was reported to be regulated by a novel transcription factor ETV1 which is unique to gastrointestinal stromal tumors (GISTs), and RNAi-mediated silencing of KCTD10 increased cell invasion, suggesting that KCTD10 function as a tumor suppressor protein [7].However, the exact functions of KCTD10 in mammalian development remain unclear.Reports showed that KCTD10 was highly expressed in human heart, skeletal muscle, and placenta, and may regulate the development of neural tube, neuroepithelium and the dorsal root ganglion in mammals [8], suggesting that this protein may play important roles in tissue development [3,6]. Formation of the vascular system is one of the earliest and most important events during embryogenesis in mammals.Among the early stages of vascular development in both the mammalian embryo and its extra-embryonic membranes, endothelial cell precursors differentiate and coalesce into a network of homogeneously sized primitive blood vessels in a process termed vasculogenesis [9].This primary vascular plexus is then remodeled by the process of angiogenesis, which involves the sprouting, branching, splitting, and differential growth of vessels in the primary plexus to form both the large and small vessels of the mature vascular system [9,10].In the mesoderm, cardiovascular system arises, pluripotent hemangioblast cells give rise to the blood islands, meanwhile the peripheral cells differentiate into endothelial cells (ECs) which later form the capillaries [11].The vasculogenesis and angiogenesis begin at E8.5 in mouse embryonic development.As blood vessels are essential for the transport of fluids, gases, nutrients, and signaling molecules between placenta and embryos, many genes' mutation related to angiogenesis causes embryonic delay or embryonic lethality between E8.5 and E11. 5. One of the most important pathways that control the vascular differentiation is Notch signaling, which is critically involved in many cellular processes including cell proliferation, survival, apoptosis, migration, invasion, angiogenesis, and metastasis [12].Functional studies showed that Notch signaling is crucial for the angiogenic growth in mice, fish, and human.In mice, the absence of Notch signaling results in defective yolk sac vascular remodeling and aberrant formation of arterial-venous circuits in the embryos, that often leads to embryonic death [13].There are four different Notch receptors in mammals, referred to as Notch1, Notch2, Notch3, and Notch4.Both Notch1 and Notch4 display prominent arterial expression [13].Mice with a dual Notch1 and Notch4 deletion show severe defects in angiogenesis: the uniform vessel networks initially form in the yolk sac but fail to properly remodel into large vessels and small capillaries [14,15].Notch2 is highly expressed in the heart myocardium [16].Mice homozygous for the Notch2 mutation died perinatally from defects in heart development including pericardial edema and myocardial wall atrophy [17].Notch3 is associated with CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), which is a rare autosomal dominant genetic disease characterized by recurrent stroke, migraine headaches, cognitive deficits, and psychiatric symptoms [18].In mammals, Hey and Hes represent the main Notch signal transducers during development [19].The combined loss of Hey1 and Hey2 leads to a lethality vascular defect that affects the placenta, yolk sac, and embryo itself, which has been attributed to impaired arterial fate determination and maturation [20]. In this study, we found the KCTD10-deficient mouse embryos showed delayed growth from E9.0, and died at approximately E10.5 due to angiogenesis defects, heart and neuron developmental failure.Further research showed that the key members in Notch signaling, such as Dll4, Notch1 and Notch4 were upregulated in KCTD10-deficient mice.Molecular biology studies showed that KCTD10 negatively regulated Notch signaling by mediating Notch1 proteolytic degradation. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "Mice of C57BL/6J, 129P strains were obtained from the Jackson Laboratory (Bar Harbor, ME).Mice were maintained on a normal 12 h/12 h light/dark cycle with regular mouse chow and water ad libitum at an AAALAC accredited specific pathogenfree facility.Animal welfare and experimental procedures were carried out strictly in accordance with the care and use of laboratory animals (National Research Council, 1996).All the animals were well regulated and animal ethics were approved in this research.All animal experiments were performed in accordance with the institutional guidelines of the Model Animal Research Center, Nanjing University, and Hunan Normal University.The University Committee on Animal Care of Nanjing University and Hunan Normal University approved the experimental protocols. ", "section_name": "Mice", "section_num": null }, { "section_content": "We used the standard BAC (Bacterial Artificial Clone) retrieval method to construct the Kctd10 flox allele [21].Briefly, a 9.6-kbDNA fragment containing the targeting region was retrieved from the BAC containing the whole genomic DNA sequence of Kctd10.The first loxP site was inserted into intron 2, and the second loxP together with neomycin-resistant gene flanked by FRT sites were inserted into intron 3. The construct was electroporated into 129 derived R1 embryonic stem (ES) cells.[22].Targeted ES cell clones were identified by PCR and southern blotting.Chimeric mice were generated by microinjection of the positive ES cells into C57BL/6J strain blastocysts.Genetic transmission was confirmed by backcrossing the chimera to C57BL/6J mice.As previously described [23], the Neo-cassette was removed by mating to FLP-ER transgenic mice (129S4/SvJaeSor-Gt(ROSA)26-Sortm1(FLP1)Dym/J, the Jackson Laboratory).To get the allele deletion of Kctd10, the floxed mice were crossed to EIIA-Cre (FVB/N-Tg(EIIa-cre)C5379Lmgd/J) transgenic mice (The Jackson Laboratory, Bar Harbor, ME, stock number #003314) [24] to remove the genomic DNA fragment between the two loxP sites that includes the 2 nd exon of Kctd10.Then we back-crossed the positive pups onto C57BL/6J mice to obtain heterozygous KCTD10 +/2 mice.The homozygous KCTD10 2/2 embryos were obtained by inter-cross of KCTD10 +/2 .The genotyping of the pups were identified by PCR analysis, all the genotyping primers and estimated size of PCR products are shown in Table 1. ", "section_name": "Generation of KCTD10 2/2 mice", "section_num": null }, { "section_content": "We amplified the coding sequence of mouse KCTD10 cDNA (GenBank Accession No. NM_026145) from mouse brain cDNA library using indicated primers as shown in Table 1.HA-KCTD10 was generated by inserting the above mouse KCTD10 cDNA into the plasmid pCMV-HA (Clontech).For KCTD10 RNA probe synthesis that used in situ hybridization, the probe fragment was digested by EcoR I from the plasmid HA-KCTD10 and inserted into the pBluescript II SK vector.Positive clones were verified by restriction enzyme digestion and sequencing.HA-cullin3 was kindly provided by Dr. Yue Xiong (University of North Carolina at Chapel Hill). ", "section_name": "Plasmid construction", "section_num": null }, { "section_content": "HUVECs (human umbilical vessel endothelial cells, Clonetics, Inc.) were grown in modified MCDB 131 medium supplemented with 12 mg/mL bovine brain extracts (BBE), 0.01 mg/mL human epidermal growth factor (hEGF), 1 mg/mL hydrocortisone, 2% FBS and 50 mg/mL gentamycin (as recommended by the HUVEC culture protocol of Clonetics).Cells were transfected with KCTD10 siRNA or negative control siRNA as detailed in Table 1 by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.Cells were harvested 24 h posttransfection. HUVECs were treated with 10 ng/mL VEGF-A 165 (Sigma) for the indicated time (0, 30, 45, 60, 90 and 120 min), or treated with different concentrations (0, 5, 10 ng/mL).After treatment, the cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS and protease inhibitors) for protein extraction.Sample proteins were separated on 10% SDS-PAGE gel and transferred onto a PVDF membrane (Bio-Rad, Richmond, CA).Then the membrane was detected by rabbit polyclonal anti-KCTD10 (Nanjing Chuanbo Biotech Co., Ltd.), anti-Notch1 (Santa Cruze, sc-9170), anti-Notch4 (Santa Cruze, sc-5594), anti-Jag1 (Santa Cruze, sc-8303), or anti-Fringe (Santa Cruze, sc-100756), anti-NICD (CST, 2421S) antibodies separately. ", "section_name": "Cell culture, siRNA transfection, VEGF induction and western blotting", "section_num": null }, { "section_content": "Total RNAs from embryos at different points were isolated using Trizol reagent (Invitrogen, Carlsbad, CA).Subsequently, the first cDNA strand was synthesized according to the manufacturer's protocol (Qiagen, Valencia, CA).The mRNA levels of KCTD10, Dll4, JAG1, Mfng, Hes1, Hey1 and GAPDH were quantified using TaqMan RT-PCR on the ABI Prism 7700 sequence detection platform.Cycle threshold (Ct) was determined in the exponential phase of the amplification curve.Human GAPDH was used as the internal control.The DDCt method was used to calculate fold changes in mRNA levels between controls and treated samples. ", "section_name": "Semiquantitative and real-time RT-PCR", "section_num": null }, { "section_content": "Embryos were fixed in 4% paraformaldehyde (Sigma) and embedded in paraffin (Sigma).Then the embryos were cut into sections of 6-mm thickness, and stained with hematoxylin and eosin (H&E, Sigma) or analyzed by immunohistochemistry using polyclonal goat anti-mouse KCTD10 antibody according to the manufacturer's instructions. For whole-mount staining of blood vessels, embryos or yolk sacs were fixed in 4% paraformaldehyde, then blocked in PBS containing 3% milk and 0.3% triton X-100, and incubated with rat monoclonal antibody against mouse PECAM-1 (BD Pharmingen) overnight at 4uC.Alexa 594-conjugated antibody (Molecular Probes, Eugene, OR) was used as a secondary antibody.After intermittent washing, the samples were mounted and analyzed under a Leica DMZRB microscope. For staining of cryosections, tissues were fixed in 4% paraformaldehyde for 2 h on ice, incubated in 20% sucrose/ PBS overnight and embedded in O.C.T. compound (Tissue-Tek, Sakura Finetek USA, Inc., Torrance, CA).Sections were then incubated with primary antibodies that diluted in PBS with 1% (v/ v) normal goat serum for 1 h and with the secondary antibodies under the same conditions.The primary antibodies used were anti-KCTD10 or anti-PECAM-1antibodies as mentioned above, while the secondary antibodies were Alexa 594 goat anti-rat and Alexa 488 goat anti-Rabbit antibodies (Molecular Probes).Hoechst 33258 (Sigma) was used to stain the nucleus.The slides were mounted with 50% glycerol in PBS and images were acquired under a fluorescence microscope (Leica). ", "section_name": "Histology and immunostaining", "section_num": null }, { "section_content": "Embryos were fixed and processed according to the previously published protocols [25] with the following modifications: Endogenous peroxidases were quenched with 6% H 2 O 2 for 2 h prior to proteinase K digestion and hybridization.Hybridization was performed for 40 h at 63uC in 56 SSC (pH 4.5), 50% formamide, 5 mM EDTA, 50 mg/mL yeast tRNA, 0.2% Tween 20, 0.5% CHAPS, and 100 mg/mL heparin.Color was developed with the NBT/BCIP substrate.Embryos were post-fixed and photographed in 50% glycerol in PBS. ", "section_name": "Whole mount in situ hybridization", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "It was previously demonstrated that KCTD10 is predominately expressed in the lung, followed by the heart and testis in the rat [3].Other groups cloned KCTD10 from a human aorta cDNA library, and their data showed a high level of KCTD10 expression in adult human heart, skeletal muscle and placenta [6].In order to explore the functions of KCTD10 during mouse early embryogenesis, whole-mount in situ hybridization was used to determine the temporal expression of KCTD10.Embryos were collected and stained from embryonic day of 8.5.At E8.5, when embryos completed the ''turning'' process that included development of onset of heart and blood vessels, KCTD10 mRNA signals mainly appeared in the heart, brain, dorsal aorta and umbilical vein (Fig. 1A).While at E9.5 and E10.5, strong KCTD10 signals were detected in the brain neuroepithelium, the optic vessel, otic vesicle, only weak signals were observed in the heart and the dorsal aorta (DA) (Figs.1B,C).In addition, sections of the brain neuroepithelium, dorsal aorta and heart of E9.5 embryos showed KCTD10 mRNA signals (Figs. 1D,E).In addition, in situ hybridization and immunohistochemistry results indicated that KCTD10 was also expressed in the somite boundaries (Figs.1B, C andF).Thus, mouse KCTD10 was mainly expressed in the dorsal aorta and heart at E8.5, and strongly expressed in brain neuroepithelium, optic vessel, and otic vesicle at E9.5 in embryos.These results indicate that KCTD10 might play crucial roles during the mouse angiogenesis and neurogenesis in early embryogenesis. ", "section_name": "KCTD10 is expressed during the early phase of mouse development", "section_num": null }, { "section_content": "The gene Kctd10 consists of seven exons, and encodes a 35 kD protein.The Kctd10 targeting constructs were designed to delete endogenous exon 2, which resulted in KCTD10 loss of function of KCTD10 (Fig. 2A).The positive ES cells and knockout mice were confirmed by Southern blotting (Figs.2B, C andD), genotyping (Fig. 2E), RT-PCR (Fig. 2F) and western blotting (Fig. 2G) analysis.The heterozygous KCTD10 +/2 mice were viable and fertile, but there were no homozygous KCTD10 2/2 mice among the off-springs.We dissected the embryos from E8.5 to E14.5, and found no detectable differences between littermates at E8.5.While at E9.5, some embryos showed developmental delay.Genotyping analysis revealed that these embryos were homozygous KCTD10 2/2 mice.Further analysis indicated that the mutant embryos showed developmental delay from E9.5, exhibited severe morphological abnormalities, and died between E10.5 and E11.5.The ratio of mutants was about 25% (Table 2), which is consistent with the Mendel's principles of inheritance. ", "section_name": "Deletion of KCTD10 is associated with embryonic lethality and growth retardation", "section_num": null }, { "section_content": "When we isolated E10.5 embryos, we found the vitelline circulation on the yolk sac of KCTD10 2/2 embryos was absent compared to that in wild type embryos (Fig. 3A).These embryos were severely retarded (Fig. 3B) and the pericardial space was enlarged (white arrows in Fig. 3A), indicating embryonic circulation defects.We then visualized the vascular network of mutant embryos and littermates by staining with an antibody against platelet endothelial cell adhesion molecule-1 (PECAM-1), a specific marker for vascular endothelial cells [26].In the mutant yolk sac, the primary vascular plexus appeared to form normally, indicating no apparent defects in vasculogenesis in the mutants.But the caliber of the major vitelline arteries and the arterial branching were reduced (Fig. 3C), suggesting that the primary vascular plexus failed to remodel and form blood vessels in the mature yolk sac.In addition, reduced tip cell formation was also observed in the abnormal yolk sac (Fig. 3D).Vascular defects were also seen in the KCTD10 2/2 embryos, among the 60% of the KCTD10 2/2 embryos, the umbilical artery (UA) became a big ball that was strongly stained by PECAM-1 (white arrow in Fig. 4A).In all of the KCTD10 2/2 embryos, the internal carotid artery (ICA), dorsal aorta (DA), arterial branches, and cardinal veins were disorganized and much thinner than those in the wild types.All these findings were confirmed by hematoxylin and eosin (H&E) staining on cross-sections.As shown in Fig. 4B, the dorsal aorta was abnormal or even missing in some severe phenotypes. We compared the dorsal aorta between E8.5 and E10.5 embryos that were immunostained by PECAM-1 (Fig. 4C).At E10.5, the dorsal aorta showed no growth in mutant embryos compared to that in E8.5 embryos (white arrows in Fig. 4C), which further confirmed the conclusion that angiogenesis defects is the direct cause for embryonic lethality.In the PECAM-1 immunostained yolk sac frozen sections, we found that the endothelial cells of the yolk sac were poorly organized in the homozygotes KCTD10 2/2 compared to those in wild type embryos (Fig. 4D).H&E staining of E10.5 yolk sac sections revealed that the abnormal yolk sac vascular surface was due to the formation of dramatically enlarged endothelial-lined lacunae between the endoderm and mesoderm layers (Fig. 4E). ", "section_name": "KCTD10 deficient embryos show angiogenic defects", "section_num": null }, { "section_content": "The disruption of KCTD10 in mice caused heart developmental failure.As described above, the mutant embryos had a dramatically enlarged pericardial edema, which was different from that in wild type littermates (Fig. 3A,B).To characterize the defects in detail, the KCTD10 2/2 and wild type embryos were sectioned and H&E stained for histological analysis.The mutant embryos showed extended pericardial edema, and the myocardial wall was relatively thinner compared with that in the wild type embryos (Fig. 5A, red arrows).In addition, KCTD10 2/2 embryos exhibited defects in cardiac valve formation.An obvious atrioventricular valve (AVV) presented in the wild type embryos, but showed defects in the KCTD10 2/2 embryonic heart (Fig. 5B, red arrow heads).Thus, KCTD10 loss of function leads to heart defects during the early embryonic development. ", "section_name": "KCTD10 deficient embryos show heart defects", "section_num": null }, { "section_content": "Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis.Most of the factors that affect embryonic vascular maturation are downstream targets of VEGF.Based on the defects in angiogenesis and heart development in KCTD10 deficient embryos, we investigated whether KCTD10 was regulated by VEGF.As described in the Materials and Methods, HUVECs were treated by VEGF-A 165 at a concentration of 10 ng/mL for different time durations (0, 30, 45, 60, 90, 120 min), the protein levels of KCTD10 were upregulated (Fig. 6A) in a time-dependent manner.As shown in Fig. 6B, the expression of KCTD10 increased when the dose of VEGF was added (0, 5, 10 ng/mL).In order to confirm the results, immunofluorescence staining was performed and enhanced protein levels of KCTD10 were detected (Fig. 6C) after treatment.The total RNA of HUVECs were extracted after 10 ng/mL VEGF treatment for indicated time, RT-PCR analysis showed the mRNA levels of KCTD10 increased(Fig.6D).These results demonstrated that KCTD10 is time-and dose-dependently regulated by VEGF. ", "section_name": "KCTD10 is induced by VEGF in a dose-and timedependent manner", "section_num": null }, { "section_content": "The phenotype of KCTD10 2/2 embryos described above were highly similar to that in Dll4 +/2 mice [27], Notch1/4 mutant mice [15] and Hey1/Hey2 double knockout mice [20].Therefore, we asked whether the disruption of KCTD10 affects the Notch signaling pathway.The total RNAs of embryos were extracted and quantitative real-time PCR (Q-RT-PCR) analysis were performed.As shown in Fig. 7A, the mRNA levels of Dll4, Fringe, Hey1, and TNFR1 robustly increased in the homozygous KCTD10 2/2 mouse embryos, whereas the mRNA levels of TNFR2 decreased.The mRNA levels of Jagged1 and Hes1 did not change.Dll4 upregulation was further confirmed by semi-quantitative reverse transcription PCR (Figs.7B,C).These data demonstrated that Dll4 is possibly involved in the KCTD10 disruption-mediated vascular defects. Furthermore, the protein levels of Notch1, Notch4, Fringe, and Jagged1 were measured by western blotting.As shown in Fig. 7D, compared to the wild types, the protein levels of Notch1, Notch4, and Fringe were up-regulated in homozygous KCTD10 2/2 embryos, while Jagged1 showed no change, which were consistent with the Q-PCR results.In addition, the protein levels of Notch1 and Fringe were also up-regulated in KCTD10 knockdown HUVECs (Fig. 7E).All these data support the hypothesis that the disruption of KCTD10 results in Dll4 up-regulation, followed by the activation of Notch signaling. ", "section_name": "KCTD10 negatively regulates Notch signaling", "section_num": null }, { "section_content": "As KCTD10 contains a conserved BTB/POZ domain and a potassium channel tetramerisation (K-tetra) domain (a relative of BTB/POZ domain) at the N-terminus.The BTB domain was known to be a highly conserved protein-protein interaction motif in multiple species.BTB-domain-containing proteins have been reported to act as substrate-specific adaptors for multimeric cullin3 ligase reactions by recruiting proteins for ubiquitination and mediating subsequent degradation of the substrates [28][29][30][31][32].We then asked whether KCTD10 induces the Notch degradation.In our study, HUVEC lysates were immunoprecipitated by rabbit polyclonal antibody against Notch1 or a negative control IgG, and the immunoprecipitates were detected by mouse monoclonal anti-cullin3 and polyclonal anti-KCTD10 antibodies, respectively.As shown in Fig. 8A, KCTD10 and cullin3 exist simultaneously in the Notch1 immune complex, but the preimmune IgG did not show any reactivity in blots.To further confirm the results, HUVECs were transfected with HA-cullin3, and the cell lysates were immunoprecipitated by antibodies against Notch1, NICD, HA, and KCTD10, immunoblotted by anti-HA and anti-KCTD10 antibody, Notch1 existed in the immune complexes of HA-cullin3 and KCTD10 and KCTD10 existed in the immune complexes of HA-cullin3.But in the immune complex of NICD (intracellular domains of Notch1), we failed to detect either KCTD10 or cullin3 (Fig. 8B).These results further confirmed the interaction between Notch1, KCTD10 and cullin3.We next wondered whether Notch1 proteins levels were affected by KCTD10.To this end, HUVECs were transfected with increasing amounts of pCMV-HA-KCTD10 (0, 0.5, 1, 2 mg).Western blotting results showed that the endogenous Notch1 protein decreased while the amounts of pCMV-HA-KCTD10 increased (Fig. 8C,D), suggesting that KCTD10 disrupted the protein stability of Notch1.Furthermore, MG132 treatment attenuated the changes(Fig.8D), and the mRNA levels of Notch1 did not affected by KCTD10 (Fig. 8E), indicating proteolytic degradation of Notch1by KCTD10.We then blocked Notch signaling activation by DAPT, which is a c-secretase inhibitor in HUVECs [27].Western blotting analysis showed that the KCTD10 protein levels increased as the DAPT concentration increased (Fig. 8F), suggesting that KCTD10 mediated the c-secretase of proteolytic processing of Notch1. In summary, KCTD10 interacts with Notch1 and Cullin3 simultaneously, and decreases the Notch1 protein stability, which indicates that KCTD10 may regulate Notch1 proteolytic degradation by interacting with cullin3. ", "section_name": "KCTD10 interacts with Notch1 and mediates its proteolytic degradation by Cullin3", "section_num": null }, { "section_content": "Recently, there are publications showed that KCTD10 plays critical roles for heart development in zebrafish [33,34].Here we explored whether KCTD10 plays critical roles in mammalian development.Like all other members in PDIP1 gene family, KCTD10 contains a BTB/POZ domain at the N-terminals, which is a highly conserved protein-protein interacting domain found in many essential transcription regulators that are involved in various developmental processes, and is important for homeostasis, cell differentiation, and even oncogenesis [35][36][37].The BTB domain is the key functional domain that directly inhibits endothelial cell tube formation and reduces VEGF expression, another BTB domain protein, PLZF, was reported to inhibit endothelial cell angiogenesis in HUVECs [38].VEGF is known as the most critical molecule controlling blood vessel morphogenesis.In our study, western blotting, RT-PCR and immunofluorescence showed that KCTD10 is VEGF-inducible in a time-and dosedependent manner, suggesting KCTD10 might be regulated by a VEGF feed-back loop.Notch signaling is known to be the downstream target of VEGF, and Dll4 expression is induced in response to VEGF [39].Dll4 is the specific mammalian endothelial ligand for autocrine endothelial Notch signaling, and is required in a dosage-sensitive manner for normal arterial patterning in development [27].Dll4 inhibits the angiogenic response of adjacent ECs to VEGF stimulation by mediating Notch signaling.Reduction of the Dll4 protein level or blocking of Notch signaling blocking enhances the formation of tip cells, resulting in dramatically increased sprouting, branching and fusion of endothelial tubes [27,[39][40][41][42][43].Heterozygous deletion of Dll4 results in prominent albeit variable defects in artery, and the down-regulation of Notch downstream target genes [44].In our study, we deleted the 2 nd exon of mouse KCTD10, which destroyed the BTB domain and caused loss of function of this gene.A homozygous deletion of KCTD10 in mice caused embryonic lethality because of cardiovascular defects, accompanied by elevated Dll4 levels and activated Fringe, Hey1, and TNFR1 in Notch signaling.These results are consistent with previous research that KCTD10 is TNF-a inducible [4], but the detailed mechanisms need to be further explored. The Notch signaling pathway is a critical component of vascular formation and morphogenesis in both development and pathology.It was reported that Notch was required for endocardial differentiation and formation of the primordial cardiac valve during the cardiac valve development [45,46].In a normal embryo, Notch signaling induces endocardial expression of the transcriptional repressor Snail, and in turn, represses VE-cadherin expression, allowing the epithelial-to-mesenchyme transformation (EMT), followed by the formation of the valvular primordium [46].Abrogation of Notch signaling in mouse or zebrafish blocks EMT [47].The Notch downstream target gene Hey was shown to control cardiomyocyte differentiation and EMT in endocardial cells [48].It is possible that deletion of KCTD10 increases Notch1 and then up-regulates Hey1, resulting in a thinner myocardium and EMT defects in the homozygous KCTD10 2/2 embryos. Notch receptors are expressed on the cell surface as heterodimeric proteins.They are composed of an extracellular domain containing up to 36 EGF-like repeats followed by 3 cysteine rich LIN repeats and an intracellular domain containing multiple protein-protein interaction domains.Notch signaling is triggered upon ligand-receptor interaction, which induces two sequential proteolytic cleavages.The first cleavage is in the extracellular domain, and is mediated by metalloproteases of the ADAM family; the second cleavage is within the transmembrane domain, and it is mediated by presenilin (PS) c-secretase activity.The secondary cleavage allows the release and translocation of Notch intracellular domain (NICD) into the nucleus [49].Consistent with reports that the KCTD10 BTB/POZ domain mediates proteinprotein interactions and is involved in proteolysis mediated by Cullin3 [11,31,[50][51][52], we revealed that KCTD10 interacts with Cullin3 and Notch1 simultaneously, and the c-secretase-specific inhibitor DAPT [27] regulates the KCTD10 protein level.The possible mechanism is that KCTD10 mediates the second proteolytic cleavage and transfers the NICD into the nucleus.Thus, KCTD10 deletion causes Notch1 accumulation, which stimulated more Dll4, and leads to disruption of angiogenesis. In summary, we generated KCTD10 knockout mice by disrupting exon 2, leading to loss of function of KCTD10.This study provided evidence that KCTD10 plays important roles in embryonic angiogenesis during mammalian development, cardiovascular development, and negative regulation of Notch signaling.Thus, KCTD10 is one of the most important factors in regulating embryonic development and angiogenesis. ", "section_name": "Discussion", "section_num": null } ]	[ { "section_content": "We thank Prof. Yang Zhong-zhou (Nanjing University) and Prof. Wu Xiushan (Hunan Normal University) for their kind suggestions. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "This work was supported by the 973 project of Ministry of Science and Technique of China (No. 2010CB529900), the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (No. 20134486), the program for excellent talents in Hunan Normal University (No. ET13107), and the Construct Program of the Key Discipline of Basic Medicine in Hunan Province.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "Conceived and designed the experiments: SX J. Zhang.Performed the experiments: KR JY MY.Analyzed the data: KR JY MY XG X. Ding J. Zhou XH JC X. Deng.Contributed reagents/materials/analysis tools: JY MY XG X. Ding J. Zhou XH JC X. Deng.Wrote the paper: KR. ", "section_name": "Author Contributions", "section_num": null } ]
10.1371/journal.pone.0073098	Single-Cell Analysis of Thymocyte Differentiation: Identification of Transcription Factor Interactions and a Major Stochastic Component in αβ-Lineage Commitment	T cell commitment and αβ/γδ lineage specification in the thymus involves interactions between many different genes. Characterization of these interactions thus requires a multiparameter analysis of individual thymocytes. We developed two efficient single-cell methods: (i) the quantitative evaluation of the co-expression levels of nine different genes, with a plating efficiency of 99-100% and a detection limit of 2 mRNA molecules/cell; and (ii) single-cell differentiation cultures, in the presence of OP9 cells transfected with the thymus Notch1 ligand DeltaL4. We show that during T cell commitment, Gata3 has a fundamental, dose-dependent role in maintaining Notch1 expression, with thymocytes becoming T-cell-committed when they co-express Notch1, Gata3 and Bc11b. Of the transcription factor expression patterns studied here, only that of Bcl11b was suggestive of a role in Pu1 down-regulation. Individual thymocytes became αβ/γδ lineage-committed at very different stages (from the TN2a stage onwards). However, 20% of TN3 cells are not αβ/γδ-lineage committed and TN4 cells comprise two main subpopulations with different degrees of maturity. The existence of a correlation between differentiation potential and expression of the pre-TCR showed that 83% of αβ-committed cells do not express the pre-TCR and revealed a major stochastic component in αβ-lineage specification.	[ { "section_content": "In the thymus, T lymphocytes develop from precursor cells that do not express CD4, CD8 or CD3.These triple-negative (TN) cells undergo several successive differentiation stages.The early thymus progenitors (ETPs) are CD44 + c-Kit + IL-7R 2 CD25 2 and are still able to generate myeloid cells, natural killer (NK) cells and rare B cells.These precursors upregulate c-Kit, IL-7R and CD25 and generate the TN2a population.The latter cells have lost B cell potential and, when compared with the ETP population, are poorly capable of generating NK cells (thus indicating significant T cell commitment).However, full T cell commitment is only achieved when TN2a thymocytes downregulate the expression of c-Kit and IL-7R to become TN2b cells.The TN2b populations then lose CD44 expression to yield TN3 thymocytes -the most abundant TN population.It is believed that the majority of TCR-l and TCR-b complete rearrangements occur during this differentiation phase.Successful rearrangements enable TN3a thymocytes to pass the pre-TCR/cd check point and become TN3b thymocytes.This selection step induces a major proliferative burst and the upregulation of CD27, which reportedly discriminates between selected and non-selected cells.The TN3b thymocytes further progress to the TN4 stage (where expression of CD25 is lost) and eventually co-express CD4 and CD8ab heterodimers to become double-positive (DP) thymocytes.It is known that all TCR-ab + CD4 + or CD8ab + thymocytes pass through an intermediate DP differentiation stage.In contrast, although the majority of cd lineage cells do not transit through a DP differentiation phase, they reportedly emerge at various differentiation stages (from TN3 through to DP thymocytes). Although T cell commitment is dependent on the master regulator Notch1, the Gata3 and Bcl11b transcription factors (TFs) must associate to Notch1 to induce this commitment [1].The lack of either Notch1 or its target gene Gata3 induces a similar, early block in TN1 cell differentiation [2,3].Investigations of Bcl11b's role have yielded contradictory results [4,5,6,7].Early studies of Bcl11b 2/2 thymocytes reported an increase in TN3 CD44 2 CD25 + thymocyte counts, massive apoptosis and elevated TCR-cd generation [7].In contrast, both Bcl11b inactivation in the fetal liver and bone marrow (BM) progenitors in culture on OP9DL1 cells induced a TN2a differentiation arrest [4,5,6], with Bcl11b-deficient precursors acquiring the selfrenewal capacities that are characteristic of stem cells.Other critical TFs downstream of Notch1 signaling (Hes1 and Tcf-1) also control early T-lineage development [8,9].However, it is not clear how these TFs interact.Although the expression of non-T master genes also declines, it is not known how this is induced or whether total repression of the non-T master genes is required for T cell commitment. T-cell-committed precursors eventually become committed to the ab or cd lineage.Despite significant research efforts, there is still much debate as to (i) the precise differentiation stages at which commitment occurs and is completed and (ii) the relative roles of a particular combination of master genes (the so-called ''stochastic model'') versus TCR-cd/pre-TCR signaling/signal strength (the so-called '' instructive model'') (reviewed in [10,11]).In support of the stochastic model, a fraction of TN2 thymocytes thought not to express either signaling complex is already lineage-committed [12] and intrathymically injected fetal TN2 IL-7R high cells generate predominantly TCR-cd cells [13].Lastly, only about half of TN2 cells express Sox13, which reportedly may be required for cd lineage commitment [14].However, the predominant generation of TCR-cd cells by TN2 IL-7R high fetal thymocytes could be specific for the fetal thymus, which exports TCR-cd cells (but not TCR-ab cells) before birth [15].Given that TCR-c rearrangements are IL-7-dependent, these TN2 cells could be a minor fetal subset already expressing the TCR-cd. The opposing view attributes lineage commitment exclusively to TCR-cd/pre-TCR-dependent signals and questions the precise differentiation stage at which final commitment occurs.Hence, thymocytes could be ''diverted'' from their lineage choice by differences in TCR signal strength, with strong TCR-cd signals inducing a cd lineage and weaker pre-TCR signals inducing ab lineage choices [16,17].Overall, the lineage choice may be flexible until TN thymocytes differentiate into either DP cells (for the ab lineage) or CD24 2 TCR + CD4 2 CD8 2 populations (for the cd lineage) [18].Lastly, it has been claimed that the presence of the pre-TCR reduces the proportion of TCR-cd + cells expressing out-of-frame TCRb rearrangementsindicating that pre-TCR signaling has deviated TN cells towards the TCR-ab lineage [19]. The debate between stochastic and instructive models has not been resolved, since methodological aspects of the studies used to support each hypothesis have been criticized.The plating efficiency of single cells in OP9DL1 differentiation cultures (used in experiments favoring the stochastic model) was low, since 50% of the TN2 thymocytes and 73% of the TN3 thymocytes did not generate progeny [12].Plating efficiencies were even lower in experiments supporting instructive models, since 96% of the plated single cells either did not grow in culture or deviated to alternative lineages [18].These low plating efficiencies cast doubt on whether these differentiation behaviors truly reflect the properties of the cell populations studied.Lastly, the evaluation of the role of pre-TCR signaling in deviating T cells towards the TCR-ab lineage could be biased, since 60% of the studied TCR-cd cells (i.e.those lacking detectable TCRB rearrangements or with only mono-allelic TCRB rearrangements) were excluded a priori as PCR failures.However, excluded samples were not tested for the presence of the b locus in a germ-line configuration and it has never shown that all TCR-cd cells have bi-allelic TCRB rearrangements. Overall, the conflicting results mentioned above highlight several of the limitations of previous experimental approaches.Gene ablation frequently has many effects on cell behavior and so it may be difficult to identify a given gene's precise role in each particular pathway/differentiation step.Since the role of each TF is known to be critically dependent on its concentration, overexpression studies may reveal roles or activate pathways that do not reflect the TF's behavior at physiological concentrations.All such studies focus on the behavior of a single gene and thus are unlikely to reflect the complexity of T cell differentiation, which depends on interactions between many genes in each individual cell.Lastly, so-called ''lineage tracers'' for the expression of both the d chain and the pTa have been developed but the results were disappointing.The lineage tracers were expressed well upstream of the expression of the respective genes [20,21] and thus failed to trace the respective lineages. These problems could be resolved by combining (i) efficient single-cell methods for the accurate quantification of gene expression and gene association with (ii) single-cell differentiation cultures with a high plating efficiency.Here, we describe our development of this type of method.We created a genetic profiling technique that simultaneously quantifies the absolute number of mRNAs for each of the nine TFs involved in thymocyte differentiation (along with Cd3e, Rag1 and pTa) in each individual cell.Importantly, all the steps in this method have been validated; the method has a plating efficiency of 99%-100% and can reliably detect as few as 2 mRNA molecules per cell [22].Furthermore, we combined this method with efficient single-cell differentiation cultures by stimulating individual precursors with the OP9 cell line transfected with the thymus Notch1 ligand DL4 [23].The use of DL4 (rather than DL1) substantially increased our plating efficiency and enabled us to characterize the kinetics of ab/cd lineage specification more accurately.Overall, the combination of these two approaches allowed us to correlate individual thymocyte differentiation potential with its co-expression of several genes.Our results reveal how different TFs associate during T cell commitment and show that 83% of ab-lineage-committed thymocytes do not express the pre-TCR. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "To study T cell commitment and ab/cd lineage specification, lineage-negative (CD8 2 CD3e 2 B220 2 Mac1 2 CD19 2 CD11c 2 NK1.1 2 CD11b 2 DX5 2 ) TN thymocyte progenitors were subdivided according to their c-Kit/CD44/CD25/CD27 profile into subpopulations with differing differentiation potentials: ETPs, TN2a and b, TN3a and b and TN4 (Fig. S1).Individual cells from each subset were sorted in order to determine (i) the proportion of cells expressing each gene of interest (i.e. the ''expression frequency''), (ii) the number of mRNA copies per cell for each expressed gene (i.e. the expression/transcription level) and (iii) the gene combinations expressed by each individual cell (i.e. gene co-expression). The approach used to determine these parameters is summarized in Fig. 1A and described in detail in the Methods section.To be used for all possible gene combinations, the approach requires strict rules for primer selection and amplification steps.We have thoroughly described and validated these rules previously [22].For each particular set of genes studied, the primers must have two additional properties.Firstly, to enable quantitative comparisons of the expression of different genes, all individual amplifications must have the same efficiency.Secondly, given that the initial PCR amplifications are performed in the presence of 18 individual primers, it is essential to show that neither the primers nor the generated amplicons compete with one another.The primer combinations used in the present experiments fulfilled both criteria; all the amplifications of individual genes had the same efficiency (as shown by the parallel slopes for each individual amplification; Fig. 1B) and did not compete with one another (as shown by similar amplification efficiencies when amplifications were performed either for a single gene or for all genes simultaneously.(Fig. 1B,C).Simultaneous compliance with these two criteria enabled us to quantify the absolute number of mRNAs coding for each gene in each individual cell, relative to a simultaneously amplified standard with a known mRNA copy number.Moreover, negative results can be accepted with confidence because by using two independent criteria, we have already demonstrated that the method can reliably detect as few as two mRNA copies per cell [22]. When interpreting the results, it is essential to understand the major differences between the novel information provided by the present approach and that obtained using gene expression arrays.Arrays are easy to perform and enable almost the entire mouse genome to be screened.In contrast, the single-cell method is laborious and only allows around 20 known genes to be screened each time.However, our present and previous results demonstrate that single-cell assays provide fundamental information that cannot be obtained in array studies, and vice-versa.By covering the entire genome, arrays have a fundamental role in identifying genes that were previously not known to be involved in a particular differentiation process.Once these genes have been identified, arrays do not provide much information on gene expression behavior and are frequently misleading, since they can only identify the average level of gene expression in a cell population.An example of this problem is shown in Fig. 1C, where the same two genes (a, b) are studied simultaneously in the same cells at the population level (100 cells together) or at the single-cell level.When studied at the population level, genes a and b appear to be expressed similarly, since their amplification curves overlap.In contrast, when studied at the single-cell level, the expression is found to be totally different: gene b is expressed moderately by all cells, whereas gene a is strongly expressed but only by 10% of the cells.This example highlights another major limitation of arrays; they cannot identify the presence of subpopulations of cells with different characteristics.Since only average gene expression is evaluated, it is not possible to determine whether an increased signal is due to (i) a higher frequency of expressing cells, (ii) the same proportion of cells expressing higher levels of the gene or (iii) a loss of expression in most cells and compensatory expression of very high levels by a small subpopulation.The same reasoning applies to a reduction in signal or to an apparent absence of change in gene expression: i.e., all possible combinations of frequency vs. expression level variations or general expression vs. restricted expression with potentially major biological significance cannot be characterized in array studies.Importantly, arrays cannot determine gene associations at the single-cell level; since most biological processes (particularly during differentiation) require the co-expression of several genes, singlecell analysis is the only method capable of identifying interactions between different molecules. Lastly, to simplify the monitoring of complex data, we divided gene expression profiles/interactions into those leading to T cell commitment and those involved in ab/cd lineage specification.The overall gene expression profiles of all thymocyte subpopulations are shown in Fig S2. ", "section_name": "Cell populations and single-cell genetic profiling", "section_num": null }, { "section_content": "Deletion of Notch1 or inhibition of Notch1 signaling in hematopoietic stem cells blocks T cell development [2].Similarly, Gata3 deletion results in the absence of mature T cells and the generation of only a small number of ETPs [3].With a view to determining why a Notch1 or Gata3 deficiency might induce similar blocks in TN cell differentiation, we studied the generation and properties of TN cells in chimeras injected with fetal liver cells from Gata3-competent, Gata3-deficient or haplo-insufficient donors (Fig. 2A).Competent cells reconstituted TN populations as found in normal thymi, whereas Gata3 2/2 precursors only generated rare CD44 + CD25 2 cells expressing low c-Kit levels [3].Furthermore, we found that Gata3 +/2 precursors had normal c-Kit expression levels but were yet poorly able to generate TN1 and TN2 populations.The low number of TN1 cells recovered from these chimeras prevented us from performing extensive single-cell studies.However, in the 27 individual Gata3 2/2 TN1 thymocytes recovered in two independent experiments, Notch1 expression frequencies were much lower (Fig. 2B).Moreover, the average Notch1 transcription rate in the rare Notch1 + cells was 115 mRNAs/cell (compared with 750 mRNAs/cell in controls).It could be argued that the Gata3-deficient TN1 thymocytes were not directly comparable to ETPs, since their c-Kit expression level is much lower.Although Gata3 +/2 TN1 cells expressed normal c-Kit levels (Fig 2A), the proportion of cells expressing Notch1 was much lower than in Gata3 +/+ cells: the Notch1 expression levels in positive cells averaged 256 copies/cell (vs.750 copies/cell in controls) (Fig 2B).In contrast, the expression level of the Notch1 cofactor RbpSuh did not change during these stages (Fig. 2C).These findings demonstrate a dose-dependent role of Gata3 in sustaining Notch1 expression.It is likely that these mutual interactions are involved in the TN1 to TN2b transition and they may explain why the ablation of the Notch1 or Gata3 gene induces a TN1 differentiation block.Gata3/Notch1 interactions may also be required for the induction of Bcl11b.This TF is detected in TN2a cells when the concentrations of both Gata3 and Notch1 peak (Fig. 2D).In contrast, the expression levels of Hes-1 and Tcf-1 do not change significantly in early TN populations (Fig. 2E).Comparison of non-T-committed TN2a cells and fully T-committed TN2b populations showed major differences in the co-expression of these TFs.Only 50% of TN2a cells co-expressed Notch1+ Gata3+ Bcl11b + TcF Graphs show triplicates of independent qRT-PCRs for each gene.Upper graphs: amplifications in which all genes were reverse-transcribed and amplified together in the first RT-PCR.Lower graphs: amplifications in which each gene was reverse-transcribed and amplified separately in the first RT-PCR.The histograms compare PCR efficiency in the two conditions.(C) Examples of differences between population-based readouts and single-cell readouts.A mature monoclonal CD8 T cell population was sorted and tested on the same day for expression of two different genes (genes a and b).Upper graphs: cells were sorted at 100 cells/well, in order to mimic population studies in which only average gene expression can be evaluated.The results demonstrate that amplifications of genes a and b have the same efficiency and that both genes are expressed to the same extent.On the basis of this data, one would conclude that the two genes are similarly expressed in this cell population.Lower graphs: each well received a single cell that was tested for the expression of genes a and b.In contrast to the population studies, these single-cell studies reveal that the respective expression levels of genes a and b genes are very different: gene b is expressed at low levels by all cells and gene a is expressed at high levels by only 10% of the cells.doi:10.1371/journal.pone.0073098.g001 ", "section_name": "Towards T cell commitment: Interactions between T master regulators", "section_num": null }, { "section_content": "T cell commitment is associated with a decline in the expression of non-T master genes.The proportion of Gfi1b + cells in the TN2b populations decreased but Gfi1b expression levels were maintained (Fig. S2C).The decline in Pu1 involved a drop in transcription frequencies, transcription rates (Fig. S2C) and protein expression (not shown).With a view to observing possible influences of T master genes on the expression of non-T master genes, we studied the co-expression of these two classes.We did not find any correlations between Notch1 (Fig. 2F) or Gata3 (not shown) co-expression or expression levels on one hand and the coexpression of Pu1 on the other.In contrast, there was a very significant inverse correlation (p,0.0005,c coefficient = 20.34) between the expression/expression levels of Bcl11b and those of Pu1 (Fig. 2F).Our results indicate that Bcl11b is the only of the nine TFs studied that can potentially be involved in the downregulation of Pu1 expression. It has been reported that Gfi1b down-regulates Gata3 expression in tumor cells [24].We did not find any evidence of this role in normal TN thymocytes, since the TN2a population which expressed the highest levels of Gata3 (Fig. 2C) also had the highest proportion of Gfi1b-expressing cells (Fig. S2C). In summary, the gene co-expression patterns and interactions described here indicate a fundamental, dose-dependent role of Gata3 in the maintenance of Notch1 expression.The interactions between Notch1 (which induces Gata3 expression [25]) and Gata3 (which has a dose-dependent effect on Notch1 expression levels) suggest that a positive feedback loop promotes and sustains T cell commitment.When individual thymocytes had become fully Tcell-committed (at the TN2b stage), they all co-expressed Notch1 + Gata3+ Bcl11b + Tcf1.This commitment did not require the extinction of non-T master genes, which only occurred in TN3 thymocytes (Fig. S2C). ", "section_name": "Interactions between T-lineage and non-T-lineage master genes", "section_num": null }, { "section_content": "To better characterize ab/cd lineage commitment, we sought to establish single-cell approaches to correlate a cell's differentiation potential with its ability to express the various components of the pre-TCR. To this end, we optimized Notch1 signaling by performing single-cell cultures in the presence of the OP9 cell line transfected with DL4 (Notch1's native ligand in the thymus) [23].Since we were plating individual cells from different TN subsets with different time courses for differentiation, each culture was checked every day for growth and small samples were taken for phenotyping.This strategy ensured that the cultures that we classified as being committed to one of the lineages never generated T cells of the other lineage at different time points during culture. When compared with single-cell cultures with OP9DL1 cells [12], single-cell differentiation in OP9-DL4 cultures yielded a major increase in plating efficiency (from 52% up to 84% for TN2 cells and from 24% up to 70% for TN3 cells (Fig. 3A) and provided new knowledge on the time course of lineage specification.Although the majority of the TN2a progeny were bipotent, 18% (on average) were ab committed.In TN2b, bipotent cell frequencies declined to 48% because the remaining cells were already committed to the ab (<42%) or cd lineage (<10%) (Fig. 3A).The transition to TN3 did not increase the proportion of cells committed to the cd lineage, whereas the percentage of TN3 cells generating exclusively TCR-ab cells rose to 72%.In contrast to previous claims that all TN3 cells are lineagecommitted [12], we found that an average of 20% of the individual TN3 thymocytes were still capable of generating both TCR-ab and TCR-cd cells.The presence of this significant, bipotent TN3 population has important implications for the interpretation of studies of signal strength in ab/cd lineage specification, as discussed below.We found that bipotent and abcommitted cells generated higher cell yields (about 2610 5 cells/ culture at day 14) than cd-committed single cell cultures did (about 2610 3 cells/culture at day 14). It is known that commitment events are frequently accompanied by proliferative bursts, as described in TN4 populations after the passage through the pre-TCR/cd check point [26].We also found that the fully T-cell-committed TN2b population had a high proportion of BrdU + cells (Fig. S3) -showing that TN populations undergo an additional, major proliferative burst associated with T lineage commitment. ", "section_name": "Towards ab/cd lineage commitment: Single-cell differentiation of TN populations in OP9DL4 cultures increases plating efficiency and reveals a new time course for lineage specification", "section_num": null }, { "section_content": "To study the role of the pre-TCR in ab lineage commitment, we investigated the putative correlation between the differentiation potential of TN single cells and the latter's expression of the various elements of the pre-TCR (Fig. 3B-D).Although 18% of TN2a cells were ab-committed, none were found to express the pre-TCR.Only 30% of the cells expressed Rag1, and Rag1 expression levels in these cells were low.TCRBDJ rearrangements were quite rare and the TCR-b chain, CD3e and pTa were not expressed (Fig. 3B-D).TN2b populations were more mature, since the frequency of Rag1 expression was substantially higher and CD3e was suddenly switched on.However, these genes were expressed at relatively low levels -explaining the very low frequency of TCRB locus rearrangements.The TCRBDJ locus was mostly in a germ-line configuration and the TCR-b chain was not expressed (Fig. 3B-D).Moreover, pTa was virtually absent, B-E: Upper graphs depict expression frequencies (the proportion of positive cells) and lower grafts show the mRNA expression level in each individual cell (represented by a dot) from three independent experiments.Expression-negative cells and cells expressing fewer than 10 mRNAs/cell are not shown.Bars represent mean expression levels.Statistical analysis was performed using Fisher's exact test for expression frequencies and a Mann-Whitney rank sum test for expression levels.Asterisks correspond to a comparison of the population of interest with the population in the previous transition stage: * p,0.05, p,0.01 and * p,0.001.F. The co-expression of the different genes was studied in forty individual cells.Each dot represents an individual cell, plotted simultaneously for the number of Pu1 mRNA molecules on the X axis and the number of mRNA molecules coded as either Notch1 (upper graph) or Bc11B on the Y axis (lower graph).The correlation between the respective expressions of each pair of genes was studied in a Goodman-Kruskal gamma test, which assesses the correlation's significance (via a p-value) and nature (via the gamma coefficient, which is negative for a negative correlation and positive for a positive correlation).A polynomial trend curve is shown for each correlation.doi:10.1371/journal.pone.0073098.g002 in agreement with a publication [27] but in contrast to the latest report by the same group [28].We conclude that although 43% of TN2b cells were committed to the ab lineage, none carried the pre-TCR. Surprisingly, a large proportion of TN3 cells could not express the pre-TCR either.Although virtually all TN3 thymocytes expressed Rag1, the latter's mRNA copy number/cell increased tenfold, CD3e became ubiquitously expressed and PTa was suddenly induced in the majority of cells (Fig. 3C), most TN3 cells had yet to complete their TCRB rearrangements.DJ germ-line bands were clearly detected (Fig. 3B), which contrasts with the situation in mature TCR-ab cells [29].Although 73% of TN3 cells were committed to the ab lineage, only 17.363% expressed the TCR-b chain and therefore had the potential to express the pre-TCR (Fig. 3D).With a view to determining the proportion of abcommitted cells expressing the pre-TCR, we correlated our frequency data (Fig. 3) with the mean proportion of each TN subpopulation (Table 1).This calculation showed that on average, 83% of ab-committed cells did not express the pre-TCR.Our data clearly show that ab-committed TN thymocytes not expressing the pre-TCR cannot be regarded as an outlier population that does not follow the general rules governing ab lineage choice.Indeed, the vast majority of ab-committed cells failed to express the pre-TCR. ", "section_name": "Quantification of thymocyte populations expressing the different components of the pre-TCR", "section_num": null }, { "section_content": "The precise point at which full ab/cd lineage commitment takes place is still subject to debate.Some researchers have suggested that when TN3 cells express CD27, they are all lineage committed and ready to pass the pre-TCR/cd check-point [30].However, these studies were performed at the population level and thus could not rule out the presence of some non-committed cells within the cell set.Indeed, some researchers have suggested that full commitment occurs much later [18]. In the TN3a-to-TN3b transition, Notch1 expression did not change (data not shown), whereas Gata3, Cd3e and Aiolos expression frequencies and/or expression levels increased and those of Rag1 and pTa declined (Fig. 4A).Surprisingly, two very different cell subsets stood out within the TN4 population (Fig. 4B).Approximately 60% of the TN4 cells had much the same characteristics as TN3a thymocytes (i.e.relatively high levels of Notch1, Rag1 and pTaexpression).In the remaining cells, Notch1 and pTa were either downregulated or not expressed (as we had also found in DP cells -data not shown), Rag1 was absent and Gata3 expression levels were upregulated.This more mature population might correspond to the TCR-cd precursors that reportedly emerge at this differentiation stage [20].However, 80% of these cells expressed Aiolos (Fig. 4B), which is supposedly a marker for ab lineage-committed cells [27].Although we lack an explanation for the clear dichotomy in TN4 populations, our results reveal a significant degree of heterogeneity in late TN differentiation. ", "section_name": "From TN3 to DP: changes induced by the transition through the pre-TCR/cd check-point and TN4 heterogeneity", "section_num": null }, { "section_content": "Several approaches have been used to study early thymocyte differentiation.Here, we developed a new ex vivo approach that enabled us to describe how TN thymocytes co-express a defined set of genes under physiological conditions. Both Notch1 and Gata3 are known to have a major role in the survival and differentiation of ETPs [25].It was shown that Notch1 induces Gata3 expression, since introduction of Gata3 before the activation of Notch1 signaling fails to promote T cell development [25].However, Gata39s role in early TN differentiation remained unclear; although the ablation of Gata3 induces a major block in TN1 differentiation, the TF was found not to be essential for the generation of thymocyte progenitors in the BM.Furthermore, Gata3 does not appear to influence thymus colonization and neither blocks ETP division nor promotes ETP apoptosis [3].We found that Notch1 expression is virtually abrogated in c-Kit low , Gata3-deficient TN1 cells and is very low in c-Kit + Gata3 haplo-insufficient ETPs.These findings indicate that the survival of ETPs and the latter's progression through the differentiation process depends on interactions between Notch1 and Gata3.Notch1 induces and regulates the expression of Gata3, which in turn has a fundamental, dose-dependent role in the maintenance of Notch1 expression levels.These interactions might create a positive feedback loop and might be required for induction of the Notch1 target gene Bcl11b.Indeed, we found that the respective mean mRNA copy number for Notch1 and Gata3 increased (by a factor of 3 and 5, respectively) in the ETP-TN2a transition (when Bcl11b is first expressed).In contrast, neither Notch1 nor Gata3 co-expression patterns are compatible with a direct, suppressive effect on the expression of non-T-lineage genes.Of the TFs studied here, only the Bcl11b expression patterns were compatible with this role (with a very marked inverse correlation with the co-expression of Pu1).Although this inverse correlation does not prove that Bcl11b down-regulates the expression of Pu1, there is other independent, supporting evidence.It has been shown that (i) Bcl11b ablation prompts the differentiation of thymocyte precursors into natural killer (NK) cells [4,5,6] and (ii) Pu1 has a major role in NK generation and differentiation [31].We confirmed that full T cell commitment occurs in the TN2b population, as reported previously [28].It is usually assumed that repression of non-T-lineage master genes must be complete before T cell commitment takes place.However, our results indicate that a mere reduction in expression of these genes is enough to allow full T cell commitment, since both Pu1 and Gfi1b are still being expressed in the T-cell-committed TN2b set.In contrast, a major difference between TN2a and TN2b relates to the co-expression of Notch1 + Gata3+ Bcl11b+ Tcf-1 in individual cells.Indeed, these four genes are co-expressed by only half the TN2a cells but by all individual T-cell-committed TN2b cells.It is possible that the coexpression of these 4 genes is required for full T cell commitment.Alternatively, the very high Gata3 expression levels found in TN2b cells may have a major role in T cell commitment, as we found in the extrathymic T-cell-committed precursors in BMderived spleen colonies or in the nude spleen [32]. Once the TN2b populations had transitioned to the TN3 stage, non-T-lineage genes were totally silenced and T-cell-specific genes were turned on sequentially: Rag1 and CD3e were turned on at the TN2b stage and then upregulated in TN3, pTa was turned on in TN3 only and the transcription rates of both Gata3 and Bcl11b declined.The study of TN single-cell differentiation potential in co-cultures with the OP9DL4 cell line enabled us to increase the plating efficiency considerably.Under these conditions, a comparison between each cell's differentiation potential and its expression of T-lineage-specific genes provided insights into the mechanism of ab/cd commitment.Firstly, by looking at the time course of events on the single-cell level, we found that lineage commitment may occur at very different phases in the TN ontogeny -ranging from TN2a to TN3 or possibly even later.Concerning mechanistic aspects, about 83% of TN cells became committed to the ab lineage before they were able to express the pre-TCR.These results do not undermine the fundamental role of the pre-TCR, which is probably expressed in these cultures before TCR-ab mature cells are generated.In fact, our results redefine the pre-TCR as a differentiation factor involved in the expansion of pre-committed thymocytes and the latter's differentiation into DP cells and TCR-ab cells.This type of role fits best with the stochastic model, which separates commitment from differentiation and features an initial commitment phase that is induced by a particular combination of master genes.Further differentiation of committed cells would then occur once ab/cd-specific differentiation factors are expressed. Although not all the plated single cells differentiated in vitro, the high observed T cell counts and ab plating efficiencies contradict this hypothesis.It cannot be held that the ab lineage-committed TN2b cells detected in clonogenic assays (84% of the TN2b set) were the progeny of precursors already expressing the pre-TCR, since none of the TN2b cells expressed TCR-band only 5% expressed low levels of pTa.Similar calculations for TN3 populations reveal that the proportion of lineage-committed cells (77%) was much higher than predicted on the basis of the proportion of precursors expressing the TCR-b chain (17%, on average). These results do not rule out a fundamental role of the pre-TCR in the generation of TCR-ab cells but do redefine the pre-TCR's role as a fundamental factor in the expansion and differentiation of pre-committed cells into TCR-ab cells.It is likely that commitment to differentiation into the cd lineage also precedes the expression of TCR-c.In fact, TCR-c rearrangements in TN2 populations are very rare (L.Peaudecerf, P. Pereira and B. Rocha, unpublished data) and it has been reported that TCR-c protein cannot be detected in TN2 cells [33]. Our data showed that commitment to ab/cd lineages precedes the expression of the TCRcd or the pre-TCR in the vast majority of thymocytes; this might appear to be incompatible with previous experiments in which signal strength was found to have a role in lineage specification.In these experiments, thymocytes from cd TCR transgenic mice were shifted to DP differentiation by a reduction in the TCR-cd signal strength [16].However, the thymocyte population comprised both committed and noncommitted cells.Under these circumstances, it is not possible to say whether changes in signal strength induced lineage commitment or, in contrast, were merely required for the differentiation of precursors into TCR-cd + cells.The results of other single-cell experiments suggested that signal strength could modify cell fate relatively late in the thymocyte ontogeny and that lineage choice was flexible until TN thymocytes had differentiated into either DP cells (for the ab lineage) or CD24 2 TCR + CD4 2 CD8 2 populations (for the cd lineage).However, only 4% of the plated single cells grew in culture and deviated to alternative lineages [18].Therefore, instructive signals were only directly effective in a very small number of TN cells.Lastly, it was also shown that the presence of the pre-TCR reduced the proportion of TCR-cd cells expressing out-of-frame TCRb rearrangements; this observation suggested that pre-TCR signaling deviated TN cells to the TCRab lineage [19].Again, both the low number of characterized cells (21 in pre-Ta-deficient mice) and the choice of selection criteria could have biased these conclusions.This evaluation assumed that all TCR-cd cells should have rearranged both TCRB alleles and thus excluded about 60% of the cells studied (i.e.those having undergone no TCRB locus rearrangements or only mono-allelic rearrangements).However, taking account of these excluded cells would considerably change the prediction of out-of-frame rearrangements.Lastly, one can argue that the differentiation potential of thymocyte precursors in OP9-DL4 cells does not correspond to lineage commitment because the latter requires the presence of other signals.In this hypothesis, commitment can only be demonstrated when none of the signals introduced into these cultures modified the fate of the plated cells.This approach confuses commitment signals with differentiation signals.In fact, later-stage signals (such as TCR-cd pre-TCR signal strength) may only be required for differentiation.For cells committed to a particular lineage, only those expressing the differentiation factor corresponding to that lineage will differentiate.Although the introduction of differentiation factors may modify the proportion of cells committed to either lineage, this change may be only reflected by a reduction in the plating efficiency.Moreover, since all TN populations studied to date have been mixtures of lineagecommitted and non-committed cells, we consider that commitment studies must be always performed at the single-cell level.Overall, the approach described here can be used to quantify the expression of the pre-TCR in non-manipulated single cells and assess the latter's differentiation potential.Given the limitations of currently available differentiation cultures (which do not enable commitment factors to be distinguished from differentiation factors), our study provides novel information on the respective impacts of stochastic and instructive signals on ab/cd lineage commitment.The literature data has been interpreted as suggesting that stochastic commitment only operates in a small proportion of thymocytes (a subpopulation of the rare TN2 set) and that the remaining cells follow instructive rules.Our present data suggest that stochastic commitment operates in the vast majority of TN cells and that only a small minority of TN thymocytes follows instructive rules. We identified the TN differentiation stages at which ab/cd lineage commitment began and at which the vast majority of TN thymocytes became lineage-committed.However, we did not identify the differentiation step at which all individual thymocytes became ab/cd lineage-committed.Our data suggests that this process takes longer than proposed previously [12].We found that 20% of the TN3 cells were still bipotent.Although it has been suggested that all TN3b thymocytes are lineage-committed [30], other data revealed the presence of TCR-cd 2 TCRb 2 cells within this population [18].Moreover, we found that TN4 populations were heterogeneous; one fraction was still expressing the high levels of Rag1, Notch1 and pTa characteristic of TN3a cells, whereas another fraction did not express Rag1 and showed the downregulation of Notch1 and pTa characteristic of DP and mature thymocytes.The latter fraction might correspond to the TCR-cd precursors that reportedly emerge at this differentiation stage [18].However, this hypothesis was contradicted by the cells' co-expression of Aiolos, which is thought to demonstrate ab commitment [30].The considerable heterogeneity seen in late TN populations indicates that further studies will be required to characterize this subset. ", "section_name": "Discussion", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "Triple-negative populations were obtained from 6-to 8-weekold B6 mice purchased from Centre d'Elevage R. Janvier (Le Genest St Isles, France).Thymocytes from Gata-3 deficient mice were obtained by injecting Ly5.2 fetal liver precursors into sublethally irradiated Ly5.1 hosts [34]. ", "section_name": "Mice", "section_num": null }, { "section_content": "All experiments were carried out in accordance with the guidelines of the French Ministry of Agriculture, under a personal license (number 75-1026).No approval was necessary (government decree number 2013-118) ", "section_name": "Ethics statement", "section_num": null }, { "section_content": "The monoclonal antibodies (mAbs) used for cell sorting were obtained from BD Pharmingen (San Diego, CA, USA).For cell sorting, Lin + thymocytes were first depleted by magnetic sorting using TER119, Gr1 (RB6-8C5) and CD8 rat mAbs, sheep anti-rat IgG-conjugated beads and sheep anti-mouse IgG-conjugated beads (Dynabeads M-450; Dynal Biotech A.S., Oslo, Norway).Triple-negative cells were further labeled with antibodies against lineage antigens (Mac-1, NK1.1, TCRab, TCRcd, CD8a and CD19), CD25, CD44, c-Kit and CD27.Single cells from each TN subpopulation were collected in individual PCR tubes (containing 5 ml of 0.1% PBS-DEPC using a FACSAria I equipped with an automatic cell deposition unit (BD Biosciences, San Diego, CA, USA)) and stored at 280uC. Intracellular staining for TCRb and CD3e proteins was performed with the Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences), according to the manufacturer's instructions.To target cells in S-phase, mice were injected intraperitoneally with BrdU (BD Biosciences) (as described previously [26]) and studied 60 minutes later.Incorporation of BrdU was performed according to the manufacturer's instructions. ", "section_name": "Antibodies", "section_num": null }, { "section_content": "These experiments were performed as described in the previously published validation study [22].Briefly, single cells from each TN subpopulation (Fig. S1) and the TN3a TN3b populations (data not shown) were collected in individual PCR tubes (containing 5 ml of 0.1% PBS-DEPC using a BD FACSAria I equipped with an automatic cell deposition unit (BD Biosciences)) and stored at 280uC until use.The cDNA was prepared by genespecific RT with the specific 39 primers for all the genes studied and for a housekeeping gene.This step was followed by a 14-cycle amplification in the presence of specific 59 primers for all genes.The PCR products from each single-cell amplification were then aliquoted into wells in which the expression of each gene was quantified separately by using semi-nested qPCRs.Slope values for the exponential PCR phase were determined using the Sequence Detector System Software (version 2.2, Applied Biosystems Inc., Branchburg, NJ, USA).The process for primer selection is indicated as indicated.Gene sequence data were taken from the Ensembl (http://www.ensembl.org)and NCBI nucleotide databases (http://www.ncbi.nlm.nih.gov/entrez/).Primers were designed manually, according to the strict rules that we have described previously.Briefly, 39 and 59 primers were chosen from different exons (in order to avoid genomic amplification).To obtain similar amplification efficiencies for all mRNAs, we designed 20-base-pair (bp) primers for similar-sized fragments and with similar melting temperatures (T m ) and guanine-cytosine contents (around 50%).To avoid primer competition, primers and potential amplicons must not cross-hybridize.Primer compatibility was first assessed using Amplify 1.2 software (http://engels.genetics.wis.edu/amplify) and then tested experimentally in the competition experiments described below.We frequently found competition that had not been predicted by the Amplify 1.2 software, and so other primer pairs had to be selected and tested.The primers used in this study are listed in Table S1.The generation of a standard cDNA with a known number of mRNA molecules is as indicated.To allow absolute quantification of the number of mRNA molecules present in each cell, we prepared a standard cDNA with a known number of mRNA molecules and that could be amplified with the same efficiency as in the other RT-PCR reactions.To this end, we used Cmyb cDNA extracted and amplified from Lin-ckit + Sca1+ (LSK) BM cells.This cDNA was harvested from a 1.5% agarose ethidium bromide gel and then purified using the Wizard SV Gel/PCR Clean-up System (Promega Corporation, Madison, WI, USA).The purified cDNA was then assayed using the Picogreen incorporation method (Molecular Probes Inc., Eugene, OR, USA), using an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems Inc.).The concentration of the purified Cmyb cDNA was then determined with respect to a dsDNA from plasmid (Molecular Probes Inc.).The validation of the different steps in the RT-qPCR reaction is as indicated.This method requires strict rules for primer selection and the simultaneous validation of several different steps, as described in detail previously [22] and below.Firstly, we had to prove that the efficiency of reverse transcription (RT) was the same for each gene studied.Secondly, primers have to be selected so that (i) they have the same efficiency and (ii) neither the primers nor the amplicons they generate compete with one another (since multiple primers are present in the first PCR reaction).While selecting primers with similar efficiency is usually an easy task, simultaneously preventing competition usually requires the selection of primers from very different parts of the gene.These different locations prevented us from using PolyA reverse transcription, which favors 39 RTs and thus could differ in its efficiency as a function of primer location.We preferred to use gene-specific RT and have already shown that the RTs of each gene had the same efficiency.Secondly, this single-cell RT-qPCR comprises two independent steps -first an RT-PCR and then a semi-nested qPCR.We thus had to determine the number of amplification cycles in first RT-PCR reaction that allowed the detection of a very low mRNA copy number without reaching saturation (even when transcription rates were very high).To evaluate this parameter, we amplified different, known numbers of synthetic RNA molecules by varying the number of amplification cycles in the first PCR.We found that the use of 14 cycles enabled us to detected as few as 10 RNA copies and did not cause saturation (even when 108 RNA molecules were amplified).However, since the amplifications of synthetic RNA and cellular RNA may differ, we further refined our evaluation of the method's sensitivity.Rather than amplifying mRNA, we looked at whether our amplification procedures could detect a given gene in each individual cell.In the absence of RT and after proteinase K treatment, our primers and amplification procedures indeed detected the gene in all single-cells [22].These data proved that the method detected a copy number as low as two, since only two copies of a gene were present in the genome.It is noteworthy that all the primers used here are located in different exons.Hence, DNA amplification could not have biased our RT-PCR results because otherwise the PCR product would have contained the intron and would have been larger than the PCR product generated after mRNA amplification.In addition to these general rules (which apply to all gene combinations studied), it was also necessary to demonstrate that all amplifications of each simultaneously studied set of genes have the same efficiency and do not compete with one another.The primers used in this study complied with both these requirements: they all had the same efficiency (as shown by the parallel amplification curves) and did not compete with one another (since amplifications using a gene's primers alone gave the same results as when the primers for all the genes were present). ", "section_name": "Quantitative single-cell RT PCR", "section_num": null }, { "section_content": "OP9 or OP9-DL4 cell lines were cultured in the presence of 1 ng ml 21 IL-7 and 5 ng ml 21 of the corresponding Flt3 ligand [12].For the mass culture assays, 200 TN cells were cultured in 24-well plates.For clonogenic assays, individual cells were cultured in a 96-well round bottom plates.To prevent artifacts associated with different growth or survival rates, individual cultures were monitored for growth and small numbers of cells were sampled to identify the progeny at different time points after plating. ", "section_name": "OP9DL4 co-cultures", "section_num": null }, { "section_content": "TCRBDJ and VDJ rearrangements were identified as described previously [35].(DOC) ", "section_name": "PCR analysis of T cell receptor gene rearrangements", "section_num": null }, { "section_content": "", "section_name": "Supporting Information", "section_num": null } ]	[ { "section_content": "We thank E. Six and I. Andre ´-Schmutz for the OP9 line transfected with DL4, R. Wolterink for Gata3 mutant mice, P Kastner and S Chan for Pu1gfp mice and J. Me ´gret for single-cell sorting.SE thanks Marie Cherrier for carefully reading the manuscript. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Unit 1020 receives basic funding from the Institut National de la Sante ´et de la Recherche Me ´dicale.AB was funded by La Ligue Contre le Cancer, the European Research Council (ERC), and the Socie ´te ´Franc ¸aise d'He ´matologie.LS was funded by the Fondation pour la Recherche Me ´dicale (FRM).LGR was funded by the Ministe `re ", "section_name": "", "section_num": "" }, { "section_content": "Conceived and designed the experiments: AB SE SC.Performed the experiments: AB LS SC VP AL MBF.Analyzed the data: BR SE SC AB.Contributed reagents/materials/analysis tools: SC LG.Wrote the paper: BR SE AB. ", "section_name": "Author Contributions", "section_num": null } ]
10.1371/journal.pgen.1002577	Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1	Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.	[ { "section_content": "Notch signaling mediates communication between neighboring cells to control cell fate decisions in all metazoans [1,2].The mammalian genome encodes four Notch receptors (Notch1-4) and at least five ligands (Jagged1, 2 and Delta-like 1, 3, 4) [3].In the canonical Notch pathway, binding of the ligands to the Notch receptors present on the neighboring cell surface triggers two successive intramembrane proteolytic cleavages of the receptors mediated by the c-secretase complex and resulting in the release of the Notch intracellular domain (NICD) [4,5,6].Upon its release from the plasma membrane, NICD translocates to the nucleus where it interacts with a transcription factor of the CSL family (RBPjk/CBF-1 in mammals) to activate transcription of target genes [7].Among the best known targets of Notch/RBPjk signaling are the Hes/Hey family of basic helix-loop-helix (bHLH) transcription repressors [8].However, the regulation of individual Hes/Hey proteins by Notch and their role in mediating Notch function are highly dependent on cell context.In addition to the canonical pathway, Notch has also been reported to signal through noncanonical, RBPjk-independent mechanisms, but the molecular nature of these mechanisms is not well understood [6,9,10,11]. Notch signaling has emerged as a critical regulator of the mammalian skeleton.Initial mouse genetic studies identified a role for Notch in axial skeletal patterning, as mice lacking either Deltalike 3 (Dll3) [12], presenilin 1 (PS1) [13,14], a catalytic subunit of the c-secretase complex, or lunatic fringe, a glycosyltransferase that modifies Notch proteins [15], exhibited defects in the axial skeleton due to deficiency in somite segmentation and maintenance.In addition, mice lacking either Notch1 and 2 specifically in the limb bud ectoderm or Jagged2 globally displayed syndactyly [16,17].Consistent with the mouse studies, human mutations in Dll3 [18] were found to cause spondylocostal dysostosis, whereas those in Notch2 [10] and Jagged1 [19,20] were responsible for Alagille syndrome. More recent mouse genetic studies have expanded our view of Notch function in the osteoblast lineage.By genetically removing both catalytic subunits of the c-secretase complex, PS1 and PS2, or both Notch1 and 2 in the embryonic limb mesenchyme, we have shown that Notch critically controls postnatal bone homeostasis: the Notch-deficient long bones exhibited excessive bone formation in adolescent mice with concomitant loss of bone marrow mesenchymal progenitors [21].Consistent with the negative role of Notch in osteoblast differentiation, Zanotti et al reported that forced-expression of NICD in osteoblastic precursors reduced osteoblast numbers and caused osteopenia [22].Conversely, forced-expression of NICD at a later stage of the osteoblast lineage led to sclerosis owing to excessive proliferation of the immature osteoblasts, highlighting stage-specific functions of constitutive Notch activation in the osteoblast lineage [23,24].The negative role of physiological Notch signaling in osteoblast differentiation uncovered in mice is congruent with the clinical findings that Notch1 haploinsufficiency causes ectopic osteoblast differentiation and calcification in the aortic valves [25,26], whereas Notch2 stabilizing mutations are responsible for the Hadju-Cheney syndrome, a disorder of severe and progressive bone loss [27,28].However, the signaling cascade through which Notch inhibits osteoblastogenesis is not yet well understood. Here we have genetically assessed the role of RBPjk and Hey proteins, known components of the Notch canonical pathway, in the regulation of osteoblastogenesis.Moreover, we have evaluated the role of NFAT in the high-bone-mass phenotype caused by RBPjk deficiency.The NFAT (nuclear factor of activated T cells) family of transcription factors (NFATc1-c4) [29] have been shown to play important roles in several skeletal cell types, including chondrocytes [30], osteoclasts [31] and osteoblasts [32,33].Our results support a model wherein canonical Notch signaling suppresses osteoblastogenesis in part through inhibition of NFATc1 transcription, therefore integrating extracellular signals with transcription factors that control osteoblast differentiation. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Previously, simultaneous removal of both Notch1 and 2 (PNN mice) from the embryonic limb mesenchyme with Prx1-Cre, which targets all of the early limb bud mesenchyme and a subset of the craniofacial mesenchyme [34], caused high bone mass due to increased osteoblast differentiation [21].To discern the individual contributions of Notch1 versus 2 in the osteogenic progenitors, we employed the same Cre-loxP strategy to delete the two receptors separately.Western analyses confirmed that Notch1 or Notch2 was efficiently deleted in the limb mesenchyme of Prx1-Cre; Notch1 f/f (PN1) or Prx1-Cre; Notch2 f/f (PN2) mice, respectively (Figure 1A).As expected from our previous study of the PNN mice, PN1 and PN2 mice were viable without gross morphological anomalies.However, X-ray radiography of the limb bones at eight weeks of age revealed a marked increase in mineral content within the trabecular region of the PN2 but not the PN1 mice, when compared with their respective littermate controls (data not shown).Three-dimensional reconstruction using micro computed tomography (mCT) of the proximal tibial trabecular region confirmed this finding (Figure 1B).In particular, PN2 mice exhibited a 130% increase in trabecular bone volume owing to increased trabeculae numbers and decreased trabeculae spacing (Table 1).The PN2 phenotype was less dramatic than that of the PNN mice [21] (Figure 1B), indicating that Notch 1 performed a discernible role in the absence of Notch 2, even though deletion of Notch 1 alone did not cause an effect.Similar to the PNN mice, the high bone mass in PN2 mice was not due to decreased total osteoclast activity, as serum CTX levels, which reflect the amount of cleaved type I collagen by osteoclasts in the whole animal, did not differ significantly from the controls (Figure 1C).In addition, osteoclast number or osteoclast surface per bone perimeter did not change in either PN1 or PN2 mice (Figure 1D-1E).Therefore, physiological signaling from Notch 2, rather than Notch 1, plays a dominant role in suppressing bone formation. ", "section_name": "Notch2 plays a dominant role in suppressing bone formation", "section_num": null }, { "section_content": "To test the hypothesis that Notch suppresses bone formation through the canonical pathway, we deleted RBPjk with Prx1-Cre from the embryonic limb mesenchyme.Western analyses confirmed that RBPjk was efficiently deleted in the tibia of the Prx1-Cre; RBPjk f/f (PRBP) mouse (Figure 2A).Moreover, Hey1 and HeyL, two Notch target genes previously identified in the PNN bones [21], were markedly reduced in the PRBP tibia (Figure 2B).The PRBP mice were born at mendelian ratio with no gross abnormalities.However, at eight weeks of age, X-ray radiography revealed that the PRBP mice contained much greater mineral content within the presumptive bone marrow cavity than the wild-type littermates (Figure 2C).mCT analysis of the proximal tibia confirmed a marked increase of bone mass in the PRBP mice (Figure 2C), as reflected in a 730%, 220% or 140% increase in BV/TV, trabeculae number or trabeculae thickness, respectively, coupled with a 70% decrease in trabeculae spacing (Table 2).Consistent with the mCT data, both H&E and picrosirius red staining of the tibia detected excessive trabecular bone occluding much of the marrow cavity of the PRBP bones (Figure 2D,2E).These analyses also revealed an abnormal elongation of the growth plate hypertrophic cartilage in the PRBP bones (Figure 2D-2E); this phenotype was similar to that previously analyzed in the PNN mice and could not be contributed to changes in osteoclast numbers at the chondro-osseous junction (Figure 3A,3B).The dramatic increase in bone mass in the PRBP mice was very similar to that seen in the presenilin 1-and 2deficient (PPS) animals, but more severe than the PNN phenotype, likely due to contributions from Notch3 and 4 in the PNN mice [21].Although the data do not exclude that RBPjk may control bone formation through a yet unknown mechanism independent of Notch, the striking similarity in the bone phenotype among the PPS, the PNN and the PRBP mice indicates that RBPjk is likely the principle mediator of physiological Notch signaling in bone. We then analyzed the cellular basis for the high bone mass in the eight-week-old PRBP mice.Tartrate-resistant acid phosphatase (TRAP) staining on tibial sections revealed a strikingly uneven distribution of osteoclasts within the trabecular bone region of the PRBP mice: whereas TRAP-positive cells were more abundant than normal within the metaphyseal region, few were detected towards the diaphysis (Figure 3A).The reason for this regional disparity is not certain at present but may be due to uneven compartmentalization of osteoclast precursors within the occluded marrow cavity.Serum CTX assay did not detect any significant difference between the PRBP and the WT littermates (Figure 3C).Further investigation of the metaphyseal region revealed that although osteoclast number per bone perimeter (No.OC./mm) was higher in the PRBP mice, the spreading of individual osteoclasts (mm/OC.)was decreased, resulting in no change in the ", "section_name": "RBPjk mediates Notch function in suppressing osteoblast differentiation", "section_num": null }, { "section_content": "Osteoporosis is a disease caused by disruption of the balance between bone formation and resorption resulting in a net loss of bone mass.Although anti-resorptive agents are the current mainstay of osteoporosis therapy, novel strategies to promote bone formation are critically needed for more effective prevention and treatment of the disease.Notch signaling, an evolutionally conserved mechanism among multi-cellular organisms, was recently shown to control bone formation and therefore represents a potential target pathway for novel bone-promoting therapeutics.In this study we elucidate the intracellular signaling mechanism through which Notch controls bone formation, providing a molecular framework that may guide future drug development. percentage of bone surface covered by osteoclasts (OC.S./B.S.) (Figure 3D).Thus, the PRBP mice possessed abundant, but apparently less functional osteoclasts within the metaphyseal trabecular bone.Real-time PCR experiments showed that the mRNA levels for both the osteoclastogenic signal Rankl and the anti-osteoclastogenic factor Opg were reduced in the PRBP bone, but the ratio of Rankl over Opg (Rankl/Opg) was 230% higher in the PRBP bone than the control (Figure 3E).Moreover, the mRNA level for M-CSF, a potent mitogen of osteoclast precursors, was 690% higher in the PRBP mice (Figure 3F).The higher level of M-CSF coupled with an increased ratio of Rankl/Opg could explain the supernumerary but dysfunctional osteoclasts populat- ing the metaphyseal trabecular bone in the PRBP mice.Overall, the high bone mass in the PRBP mice was not caused by an overall decrease in bone resorption. Having ruled out resorption deficiency as the main cause for the high bone mass in PRBP mice, we next focused on bone formation parameters.Static histomorphometry of tibial sections from the eight-week-old PRBP mice revealed a marked increase in the number of cuboidal (active) osteoblasts, when normalized to either bone perimeter (60% increase) or trabecular bone area (400% increase) (Figure 4A).The number of flat (inactive) osteoblasts, when normalized to trabecular bone area, was also increased by 100% in the PRBP mice.Consistent with the increase in osteoblast number, real-time PCR experiments showed that a number of common osteoblast markers were upregulated in bone total RNA (Figure 4B).Dynamic histomorphometry showed that the mineral apposition rate (MAR), which measured osteoblast activity, did not differ significantly between PRBP and the control littermates (Figure 4C).However, the percentage of double-labeled trabcular bone surface was increased by 230% in the PRBP mice, resulting in a significant increase in the bone formation rate (BFR) within the trabecular region (Figure 4C).Thus, the increase in bone mass in the PRBP mice was primarily due to a marked increase in osteoblast numbers. To explore the mechanism responsible for the increase in osteoblast numbers, we assessed the status of apoptosis and proliferation of osteoblasts in PRBP versus wild-type bones.To this end, osteoblast protein extracts were prepared from the bone surface of the long bones, and subjected to Western analyses for activated caspase 3 and PCNA, markers for apoptosis and cell proliferation, respectively.These assays did not detect a significant difference in either protein between the genotypes (Figure 4D).Therefore, the increase in osteoblast numbers is unlikely to be caused by changes in apoptosis or proliferation, but rather due to enhanced differentiation from the progenitors. ", "section_name": "Author Summary", "section_num": null }, { "section_content": "Uncontrolled osteoblast differentiation may lead to loss of bone marrow mesenchymal progenitors and rapid age-dependent boss loss [21].To test whether this is the case in the PRBP mice, we analyzed bone mass by X-ray (data not shown) and mCT at 26 weeks of age.Indeed, bone mass was drastically reduced in the PRBP mice at 26 weeks when compared with 8 weeks (Figure 5A).When quantified, the trabecular bone mass of the PRBP tibia was no longer significantly different from the wild type at 26 weeks, representing a drastic decline from a level 730% above normal at 8 weeks (Table 2).Similarly, both trabeculae thickness and number were reduced to levels either equivalent or close to the wild type.Interestingly, bone resorption, as measured by serum CTX assays, was significantly higher in the PRBP mice over the control at 26 weeks, likely contributing to the rapid bone loss (Figure 5B).Thus,similar to the PNN mice [21], the PRBP mice, despite their high bone mass when young, rapidly lost bone with age. We next assessed the status of the mesenchymal progenitors in the bone marrow.To this end, bone marrow stromal cells (BMSC) isolated from PRBP versus wild type littermates were subjected to CFU-F (colony forming unit-fibroblast) assays.These assays were not feasible with adolescent PRBP mice due to the occlusion of the marrow cavity, and therefore performed only after six months of age.Remarkably, no type I CFU-Fs could be detected from the PRBP bone marrow at either 26 weeks (data not shown) or one year (Figure 5C), indicating a severe diminution of the mesenchymal progenitor pool.Moreover, BMSC isolated from the PRBP bone were severely deficient in undergoing osteoblast differentiation when cultured in osteogenic media and monitored by the expression of alkaline phosphatase (AP) (Figure 5D).Therefore, the PRBP animals exhibited a marked deficiency in the bone marrow mesenchymal progenitor pool. ", "section_name": "RBPjk deletion causes diminution of bone marrow mesenchymal progenitor pool and rapid age-dependent bone loss", "section_num": null }, { "section_content": "To delineate potential stage-specific requirement of Notch-RBPjk signaling during osteoblast differentiation, we deleted RBPjk with either Osx-GFP::Cre [35] or 2.3ColI-Cre [36], which are believed to target progressively more mature osteoblastic cells.Western analyses confirmed that both Cre lines efficiently deleted RBPjk in the long bones (Figure 6A).However, when assessed by either X-ray radiography or mCT, neither deletion caused any significant changes in bone mass at either 8 or 21 weeks of age (Figure 6B, 6C), a finding confirmed by quantitative analyses (Table 3).Because previous studies have suggested that Notch signaling in the more mature osteoblastic cells regulated osteoclastogenesis through modulation of Rankl and Opg [23,37], we examined osteoclasts in both the Osx-GFP::Cre; RBPjk f/f (OsxRBP) and the 2.3ColI-Cre; RBPjk f/f (ColIRBP) mice.However, serum CTX assays detected no significant changes in either OsxRBP or ColIRBP mice over controls at either 8 or 21 weeks of age (Figure 6D,6E), indicating largely normal bone resorption in these animals.Similarly, osteoclast number and osteoclast surface per bone surface were comparable between the mutant strains and their wildtype littermates (Figure 6D,6E).Thus, RBPjk does not appear to play a major role in the more committed osteoblast-lineage cells. ", "section_name": "RBPjk is not critical for later stages of osteoblast lineage", "section_num": null }, { "section_content": "To assess the role of Hey1 and HeyL in bone formation, we analyzed the bones of mice wherein the two genes have been deleted.Because previous work by others revealed no major bone phenotype in the Hey1 2/2 mice [38], we focused on the HeyL 2/2 and the Hey1/HeyL double mutant animals, all in the C57BL6 background.As Hey1 2/2 ; HeyL 2/2 mice died prematurely due to heart defects [39], we analyzed the bones of the viable HeyL 2/2 ; Hey1 +/2 animals.mCT analyses showed that the HeyL 2/2 and the HeyL 2/2 ; Hey1 +/2 mice possessed progressively more trabecular bone than their wild-type littermates at 8 weeks of age (Figure 7A).In particular, the femoral trabecular bone mass was increased by 80% and 150% over the control in the HeyL 2/2 and the HeyL 2/2 ; Hey1 +/2 animals, respectively (Table 4).Moreover, like the PRBP bones, the HeyL 2/2 ; Hey1 +/2 samples exhibited a significant increase in trabeculae number and thickness with a corresponding decrease in trabeculae spacing.At the cellular level, the HeyL 2/2 ; Hey1 +/2 bones exhibited more cuboidal osteoblasts than the wild type whereas their number of osteoclasts appeared to be normal (Figure 7B,7C).Thus, Hey1 and HeyL, like Notch and RBPjk, negatively regulate osteoblast numbers. ", "section_name": "Hey1 and HeyL mediate Notch-RBPjk signaling in osteoblast lineage", "section_num": null }, { "section_content": "We next investigated the mechanism through which Notch-RBPjk-Hey signaling regulates osteoblast differentiation.In a separate effort to identify Hey1 and HeyL target genes, we performed genome-wide ChIP-seq (Chromatin immunoprecipitation followed by high-throughput sequencing) experiments by expressing Flag-tagged Hey1 or HeyL in HEK293 cells.We identified strong binding for both proteins around the P1 promoter of NFATc1 (Figure 8A and data not shown).Importantly, Hey1 was also found to bind to the NFATc1 P1 promoter region in ST2 cells, a mouse bone marrow stromal cell line that can be induced to differentiate into osteoblasts (Figure 8B); the binding is consistent with the presence of a predicted Hey1 binding site ''CGCGCG'' within the region.In contrast, no binding was detected for the alternative P2 promoter (Figure 8B).We next focused on the functional relevance of Hey1 binding.Full-length Hey1, but not a form missing the HLH domain, suppressed the activity of the NFATc1 P1 promoter in transient transfection assays in both HEK293T and ST2 cells (Figure 8C).Because NFATc1 was previously shown to increase osteoblast numbers [33], we explored the potential involvement of NFATc1 in Notch-RBPjk signaling in bone.Real-time PCR revealed that NFATc1 mRNA was increased by 200% in the PRBP tibia over the control (Figure 8D).Western analyses identified a 70 kD isoform of NFATc1 greatly induced in the PRBP bones, whereas a 77 kD form was less affected (Figure 8E).To gain insight on the induced isoform, we employed semi-quantitative RT-PCR to identify the specific NFATc1 mRNA variant(s) increased in the PRBP bones.The NFATc1 mRNA variants are known to differ both at the 59 end containing either exon1 or 2, and at the 39 end that either terminates with exon 9b, or contains exon 9a through exon 11.By using primer pairs spanning exons 1 and 3, 2 and 3, 8 and 9b or 8 and 11, we observed a marked increase of exon 1 in the PRBP samples, whereas exon 2 was unchanged (Figure 8F).Moreover, exon 9b was enriched in PRBP over wild type, whereas exon 11 was not detectable in either genotype (Figure 8F, and data not shown).Thus, an NFATc1 mRNA variant transcribed from the P1 promoter and containing exons 1 and 9b was specifically induced in the PRBP bones. The finding above raises the possibility that the increase in NFATc1 might contribute to the high bone mass in the PRBP mice, and further that inhibition of NFATc1 activity may be able to alleviate the phenotype.To test this hypothesis, we injected littermate PRBP animals daily with either FK506, a potent inhibitor of NFAT signaling, or vehicle, for one month starting at one month of age.As expected, the vehicle treatment did not alter the high bone mass phenotype of the PRBP animals.However, FK506 markedly reduced bone mass in the PRBP mice, especially in the femur (Figure 8G and 8H, Table 5).This reversal of the high bone mass occurred in the face of decreased bone resorption in the FK506-treated animals, as indicated by a significantly lower serum CTX level (Figure 8I), presumably due to the known role of NFAT in osteoclastogenesis [31].The suppression of bone resorption may explain the observation that FK506 did not consistently correct the high bone mass in the tibia (data not shown).However, in one case where the serum CTX level was less affected by FK506, both the tibia and the femur were corrected (Figure S1).As controls, the wild-type littermates were subjected to the same inhibitor or vehicle treatment.Similar to a previous report [32], the trabecular bone mass was reduced in the wild-type animals by FK506, but the extent of reduction was modest compared to that seen in the PRBP mice (Figure S2).Overall, the results showed that NFAT inhibition could override the effect of RBPjk deletion on bone mass.Furthermore, because Hey1 directly inhibits NFATc1 expression, Notch-RBPjk-Hey signaling appears to inhibit bone formation in part by down-regulating NFATc1. ", "section_name": "NFATc1 functions downstream of Notch-RBPjk-Hey signaling", "section_num": null }, { "section_content": "The present study establishes canonical Notch signaling as a critical mechanism for maintaining bone homeostasis under normal physiological conditions.In this capacity, Notch appears to function as a gatekeeper to ensure that a proper number of [21] and NFATc1 expression (this study) (Figure 8J). The current study not only demonstrates the stage-and receptor-specificity of Notch signaling during osteoblast differentiation, but also sheds light on the intracellular mechanism mediating Notch function.Although RBPjk was previously shown to mediate the effect of NICD overexpression on both chondrogenesis and preosteoblast proliferation [24,40], this study establishes for the first time the importance of RBPjk in physiological Notch signaling within the osteoblast lineage.Moreover, the present study uncovers a direct regulation of NFATc1 expression by Notch signaling. The relationship between Notch and NFAT signaling warrants further investigation.The direct suppression of NFATc1 promoter by Hey1, and the dominant effect of FK506 over RBPjk removal support the model wherein NFAT functions downstream of and opposite to Notch signaling in regulating bone formation.However, we cannot rule out that FK506 may have NFAT- independent functions, or that the systemically-delivered FK506 acted on other cell types to affect bone mass indirectly.Our finding that Hey1 binds to and suppresses the NFATc1 promoter is consistent with a recent report that Notch inhibited NFATc1 transcription in ST2 and primary osteoblasts [41].However, NFATc1 is unlikely to be the sole effector, as simultaneous removal of NFATc1 and RBPjk with Prx1-Cre did not rescue the high bone mass phenotype caused by RBPjk deletion (data not shown).NFATc2 may play a redundant role as it was previously shown to stimulate osteoblast differentiation [32].Indeed, Western blotting showed that several isoforms of NFATc2 were markedly increased in the PRBP bones over the control (Figure S3).The mechanism for this upregulation however, is currently unknown.Future experiments with simultaneous deletion of NFATc1 and Table 4. mCT analyses of HeyL 2/2 ; Hey1 +/2 and HeyL NFATc2 will test the hypothesis that the two proteins redundantly mediate Notch function in bone.Notch-RBPjk removal from the early limb mesenchyme led to a severe deficit in type I CFU-F in the postnatal bone marrow.This phenotype could reflect either a direct requirement of Notch signaling in the CFU-F cells, or a secondary effect due to changes in osteoblast differentiation.To distinguish these possibilities, we performed lineage-tracing experiments with the Rosa26 reporter mouse, and observed that the type I CFU-F cells were not targeted by Prx1-Cre although some stromal cells in the same culture were (data not shown).In addition, experiments with a transgenic mouse (TNR) that reports Notch-RBPjk signaling [42] revealed that some stromal cells but not the type I CFU-F cells exhibited canonical Notch signaling in vitro (data not shown).Thus, diminution of bone marrow mesenchymal progenitors, as reflected by CFU-F assays in vitro, was likely to be secondary to changes in osteoblast differentiation.In this scenario, assuming mesenchymal progenitors normally exist in equilibrium with Runx2-positive osteogenic precursors, we envision that unchecked differentiation of the latter due to Notch deficiency may lead to exodus of cells from the mesenchymal progenitor pool.Alternatively, the altered bone marrow environment due to the excessive bone mass in Notch-deficient mice may be unfavorable for either establishing or maintaining a normal mesenchymal progenitor pool. A mechanistic understanding of the dominance of Notch2 over Notch1 awaits further studies.A similar dominance of Notch2 over 1 was observed during nephron formation in the mouse embryo [43,44], whereas a dominance of Notch1 over 2 was reported in the skin [45,46] as well as in osteoclasts [37].The mechanism underlying the differential roles among Notch paralogs is currently unclear, but it could reflect differences in either expression levels or ligand-binding affinities among the different receptors within a given cell type.This model predicts that Notch2 is normally preferably activated in the osteogenic progenitors.Alternatively, Notch1 and2 may be similarly activated but Notch2-NICD is more potent than Notch1-NICD in suppressing osteoblast differentiation.However, Notch 1 deficiency was sufficient to cause ectopic ossification in human aortic valves [25].Thus, the relative contribution of Notch1 versus 2 in suppressing the osteogenic program appears to be context dependent. The effect of Notch signaling in osteoblast-lineage cells on osteoclast differentiation is likely to be complex.Although TRAPpositive osteoclasts were more abundant than normal within the metaphyseal trabecular region of the PRBP bones, the total bone resorption activity was relatively normal at 8 weeks of age.However, by 26 weeks bone resorption was more robust in the PRBP mice than the littermate control, and likely contributed to the rapid bone loss seen at this age.The mechanism for the age-dependent bone resorption phenotype is not understood at present, but likely involves additional factors beyond Notch deficiency in the osteoblast lineage.In addition, unlike the PRBP mice, the PN1, PN2, OsxRBP and ColIRBP mice did not display an obvious bone resorption phenotype at either 8 or 21 weeks, even though certain changes in M-CSF, Rankl and Opg were observed in these animals (Figure S4).The lack of bone resorption phenotype in the ColIRBP mice appears to be at odds with the previous report that deletion of presenilin 1 and 2 by 2.3Col1-Cre caused an increase in bone resorption at six months, although not at three months of age [23].Besides trivial explanations such as slight age differences or genetic background variations between the two studies, the discrepancy could indicate that the previously observed effect was independent of RBPjk.In summary, the predominant function of physiologic canonical Notch signaling in the osteoblast lineage is suppression of osteoblastogenesis from the precursors.Thus, potential pharma- ceutical inhibition of this pathway in osteogenic progenitors may be beneficial for bone formation. ", "section_name": "Discussion", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "The N1 f/f [47], N2 ", "section_name": "Mouse strains", "section_num": null }, { "section_content": "Radiographs of mouse skeleton were generated using a Faxitron X-ray system (Faxitron X-ray Corp) with 20-second exposure under 25 kV.Micro computed tomography (mCT 40, Scanco Medical AG) was used for three-dimensional reconstruction, and quantification of bone parameters (threshold set at 200).Serum CTX assays were conducted with mice without feeding for 6 hours with the RatLaps ELISA kit (Immunodiagnostic Systems Ltd.).H&E, TRAP and picro-sirius red staining were performed on paraffin sections, following decalcification for postnatal samples.For dynamic histomorphometry of postnatal mice, calcein (Sigma) was injected intraparitoneally at 7.5 mg/kg on days 7 and 2 prior to sacrifice, and bones were sectioned in methyl-methacrylate.Bioquant II was used for quantification in both static and dynamic bone histomophometry.FK506 (Sigma) was dissolved in DMSO and was injected subcutaneously into one-month-old mice at 0.30 mg/kg/day for one month before harvest. ", "section_name": "Analyses of mice", "section_num": null }, { "section_content": "The CFU-F and osteoblast differentiation assays were preformed as previously described [21].Only type I CFU-Fs were scored in the present study. Transient transfections were performed as follows.ST2 were plated at 3610 4 /well in a 24-well plate overnight, and transfected with pCS2-Hey1, pCS2-Hey1-DHLH or empty pCS2 vector (0.2 mg) [21], pNFATc1-0.8P1(0.1 mg) [52] and pRL-Renilla (0.01 mg, Promega) for 8 h using Lipofectamine (1 ml/well).HEK293T cells were plated at 4610 5 /well in a 12-well plate overnight, transfected with pCS2-Hey1, pCS2-Hey1-DHLH or empty pCS2 vector (0.4 mg), pNFATc1-0.8P1(0.2 mg) and pRL-Renilla (0.02 mg) using Fugene (1.8 ml/well).The transfected ST2 or HEK293T cells were harvested at 48 hours after the beginning of transfection and subjected to dual luciferase activity assays (Promega). Western analyses were performed with bone proteins extracted with RIPA buffer from tibiae and femora that were cut into small pieces after bone marrow cells were flushed out.The Notch 1 monoclonal antibody mN1A was as previously described [53], and the Notch 2 antibody (C651.6DbHN) was from Developmental Studies Hybridoma Bank at the University of Iowa.The antibody against RBPjk was from Cosmobio (Japan). Real time PCR was performed with SYBR-Green (Roche) in ABI-7500 (Applied Biosystems) using cDNA reverse-transcribed from bone total RNA, extracted with Trizol (Invitrogen) from pulverized tibia and femur after removal of the bone marrow.Sequence information for the real-time PCR primers is listed in Table S1.The exon-specific primers for NFATc1 (Table S2) were as previously described [54], but the exons were renumbered according to the current NCBI nucleotide database.Semiquantitative RT-PCR for NFATc1 was performed at an annealing temperature of 57uC for 40 cycles.GAPDH used as loading control was amplified for 30 cycles. ", "section_name": "Cell cultures, transfections, and analyses of protein and RNA", "section_num": null }, { "section_content": "ST2 cells were infected with lentivirus to express a doxycyclineinducible Flag-Hey1 transgene.Flag-Hey1 expression was induced with100 ng/mL Doxycycline (Sigma D9891) for 12 hours.Chromatin and protein complexes were crosslinked for 10 minutes in 1% formaldehyde and flash frozen.The chromatin was sonicated to an average size of 200-450 bp using a Sonics Vibracell sonicator (model Vcx 500).Chromatin complexes were immunoprecipitated using an anti-Flag antibody (Sigma F1804).Immunoprecipitated DNA fragments were amplified by PCR using primers adjacent to a predicted Hey1-binding site within the P1 promoter of the mouse NFATc1 gene (59 -TCTCGGTCTCACTCTGACGCA -39 and 59 -TTCCCTCTTGTACACCTTTGCCCA -39), or primers near the P2 promoter approximately 4 Kb downstream (59 -TCCGGGTTTACATAAACAAGCGGC -39 and 59 -ACTG-CACACCACGCTGAACAGGAA -39). ", "section_name": "ChIP experiment", "section_num": null }, { "section_content": "HEK293 cells that express a doxycycline-regulated Flag-Hey1 or -HeyL transgene were used for ChIP-seq analysis.Cells were induced with 50 ng/ml doxycycline for 48 hours to ensure a lowlevel expression and Hey1-or HeyL-containing chromatin was immunoprecipitated with a Flag antibody.Cells carrying the same transgenes but grown without doxycycline were used as control.Preparation of ChIP libraries, Illumina sequencing and data analysis were performed as previously described [55]. ", "section_name": "ChIP-seq experiment", "section_num": null } ]	[ { "section_content": "We thank Dr. Jenna Regan for her help in surveying potential target tissues of Prx1-Cre. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "This work was supported by NIH grant AR055923 (FL) and DFG grants Ge539/11 and SFB688/A16 (MG).The bone histomorphometry studies were supported by the Washington University Center for Musculoskeletal Research (P30AR057235).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "", "section_name": "Supporting Information", "section_num": null }, { "section_content": "", "section_name": "Author Contributions", "section_num": null } ]
10.1186/s13045-014-0079-z	Potential therapeutic role of antagomiR17 for the treatment of chronic lymphocytic leukemia	Recently it was reported that microRNA from the miR-17 ~ 92 family may have a key role in chronic lymphocytic leukemia (CLL). Here, we designed specific oligonucleotides to target endogenous miR-17 (antagomiR17). In-vitro administration of antagomiR17 effectively reduced miR-17 expression and the proliferation of CLL-like MEC-1 cells. When injected in-vivo in tumor generated by the MEC-1 cells in SCID mice, antagomiR17 dramatically reduced tumor growth and significantly increase survival. Altogether, our results provide the rationale for the use of antagomiR17 as a novel potential therapeutic tool in CLL and in other lymphoproliferative disorders where miR-17 has a driver role in tumor progression.	[ { "section_content": "We have recently reported that microRNA from the miR-17 ~92 family may be responsible for the increased proliferation/survival in chronic lymphocytic leukemia (CLL) cells expressing unmutated (UM) IGHV genes and with high level of ZAP-70 [1].In particular, the enforced expression of miR-17 reduced the expression of the tumor suppressor genes E2F5, TP53INP1, TRIM8 and ZBTB4, and protected CLL cells from apoptosis [1].Here, we provide evidences that the abrogation of miR-17 expression by a specific antagomiR is sufficient to inhibit leukemic growth and progression both in-vitro and in-vivo. Peripheral blood samples from CLL patients were obtained in accordance with local Institutional Review Board requirements and declaration of Helsinki.CLL cell stimulation, microRNA and gene expression were performed as reported [1,2].MEC-1 CLL-like cell line was transfected with a molecule against miR-17 (hereafter antagomiR17), or scrambled control.In in-vivo experiments, tumors generated by MEC-1 cells into severe combined immunodeficiency (SCID) mice were treated with antagomiR17, scrambled control, or saline solution (see Additional file 1). The MEC-1 cell line expressed miR-17 levels comparable to those of CLL samples in which proliferation is triggered by CpG-ODN (Figure 1a).In MEC-1 cells, antagomiR17 transfection significantly reduced miR-17 expression respect to scrambled control, both at day 2 (mean fold change 0.84 ± 0.06; P = 0.049) and at day 4 (mean fold change 0.48 ± 0.14; P = 0.021; Figure 1b).Moreover, the TP53INP1, TRIM8 and ZBTB4 expression showed a significant up-regulation after antagomiR17 treatment both at transcript and protein levels (Figure 1c,d).Finally, MEC-1 cells showed a significant reduction (P = 0.033) of cell rate proliferation when transfected with antagomiR17 (Figure 1e).Complementary experiments performed using sorting procedures after transfecting MEC-1 cells with a Cy3-labelled antag-omiR17 (Cy3-antagomiR17, Additional file 2: Figure S1a) showed that the Cy3-antagomiR17 bright fraction presented a significant decrease in cell proliferation respect to the Cy3-antagomiR17 dim fraction at day 7 (P = 0.008; Additional file 2: Figure S1b).Notably, using a Cy3-labelled scrambled control no difference in MEC-1 cell proliferation was observed (Additional file 2: Figure S1c,d).Altogether, these data demonstrated that antagomiR17 administration effectively reduced the expression of miR-17 and cell proliferation.Tumors generated by MEC-1 cells injected into SCID mice were treated three times (day 1-8-15) either with antagomiR17 or scrambled control.AntagomiR17 dramatically inhibited tumor growth; this effect, already relevant after the first week of therapy, was maintained till the end of the treatment (Figure 2a) leading to complete regression of the mass in 1/5 (20%) of cases (not shown).Conversely, administration of the scrambled control resulted in a tumor growth kinetic superimposable to saline-treated tumors (Figure 2a).Of note, a single injection of antagomiR17 was sufficient to significantly reduce tumor growth for at least two weeks after treatment (Additional file 2: Figure S1e).Consistently, median overall survival (OS) of mice treated with antago-miR17 was significantly longer than median OS of mice treated with scrambled control (91 versus 52 days, respectively, P = 0.0018) or saline solution (91 versus 51 days, respectively, P = 0.0044) (Figure 2b).Notably, none of the mice showed signs of toxicity.Altogether, these results demonstrate that in-vivo treatment with anta-gomiR17 significantly abolishes tumor growth and increases survival. Evidences reported here underline that miR-17 knockdown is sufficient to block CLL-like cells proliferation both in-vitro and in-vivo.Clinically, despite recent treatment advances, some CLL seem to be refractory to the new drugs [3][4][5].In this context, antagomiR treatment may represent a commendable alternative, also considering recent antagomiR phase II trials [6][7][8].This strategy could be extended to other lymphoproliferative disorders where miR-17 ~92 amplification and/or overexpression have a pathogenetic role [9,10].In conclusion, our results highlight the therapeutic potential of antagomiR17, providing the rationale for its use also in the context of specific target delivering systems (e.g.nanoparticles). ", "section_name": "Findings", "section_num": null } ]	[ { "section_content": "Supported in part by: Ministero della Salute (Ricerca Finalizzata I.R.C.C.S., \"Alleanza Contro il Cancro\"; Rete Nazionale Bio-Informatica Oncologica/RN-BIO; Progetto Giovani Ricercatori n.GR-2011-02347441, n.GR-2009-1475467, n.GR-2008-1138053, Ministero della Salute, Rome, Italy; Fondazione Internazionale di Ricerca in Medicina Sperimentale (FIRMS); Associazione Italiana contro le Leucemie, linfomi e mielomi (AIL), Venezia Section, Pramaggiore Group, Italy; Ricerca Scientifica Applicata, Regione Friuli Venezia Giulia (\"Linfonet\" Project), Trieste, Italy; the Associazione Italiana Ricerca Cancro (AIRC), Grants n.IG-13227, MFAG-10327, Milan, Italy; \"5x1000 Intramural Program\", Centro di Riferimento Oncologico, Aviano, Italy. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Additional file 1: Supplemental material and methods. Additional file 2: Figure S1.In-vitro control experiments. The authors declare no competing financial interests. Authors' contributions SD, PM performed research, and contributed to write the manuscript; TDA, NM, MDB, SC, AZ, ET performed research and in vivo experiments; GDP provided patients data; SZ, in vivo experiments; VG, RB designed the study and wrote the manuscript.All authors read and approved the final manuscript. Sara Dereani and Paolo Macor contributed to this study as first authors.Valter Gattei and Riccardo Bomben equally contributed to this study as senior authors. ", "section_name": "Additional files", "section_num": null }, { "section_content": "Additional file 1: Supplemental material and methods. Additional file 2: Figure S1.In-vitro control experiments. ", "section_name": "Additional files", "section_num": null }, { "section_content": "The authors declare no competing financial interests. Authors' contributions SD, PM performed research, and contributed to write the manuscript; TDA, NM, MDB, SC, AZ, ET performed research and in vivo experiments; GDP provided patients data; SZ, in vivo experiments; VG, RB designed the study and wrote the manuscript.All authors read and approved the final manuscript. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Sara Dereani and Paolo Macor contributed to this study as first authors.Valter Gattei and Riccardo Bomben equally contributed to this study as senior authors. ", "section_name": "Authors' information", "section_num": null } ]
10.1186/s12245-024-00734-x	Manual flagging failed to identify pseudohyperkalemia in acute myeloid leukemia: case report	<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Pseudohyperkalemia is well known in acute or chronic lymphocytic leukemia, but it is very rare in acute myeloid leukemia (AML). The lab flagging system for leukocytosis to prevent pseudohyperkalemia may not work.</jats:p> </jats:sec><jats:sec> <jats:title>Case presentation</jats:title> <jats:p>A 55 year-old white man with AML was sent to emergency department for transfusion due to severe anemia. Blood test showed severe leukocytosis and elevated potassium. Repeated blood test showed his potassium was even higher. Anti-hyperkalemic medical treatment was given. He was then diagnosed with pseudohyperkalema.</jats:p> </jats:sec><jats:sec> <jats:title>Investigation</jats:title> <jats:p>I was repeatedly reassured that the lab’s manual flagging system for leukocytosis was the key in reaching the correct diagnosis. My persistent inquiries, however, revealed that the flagging system was not functioning in the care of this patient. It was clinicians’ suspicion of pseudohyperkalema that led to the correct diagnosis, although the clinicians’ recommendation of obtaining a heparinized plasma for test did not play a role because all blood samples were already heparinized. The cause of pseudohyperkalemia was pneumatic tube transport. After this incident, our laboratory is investigating the options of using the Laboratory Information System to automatically flag the results and Clinical Laboratory Scientists to make the chemistry team more aware of potentially erroneous potassium results due to pseudohyperkalemia.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Pseudohyperkalemia associated with leukocytosis still occurs. This is the first case of pneumatic tube transport causing pseudohyperkalemia associated with AML. When significant leukocytosis, thrombocytosis, hyperproteinemia, or hyperlipidemia is present, whole blood should be utilized for potassium measurements and walked to the lab instead of sent by pneumatic tube transport. Even in a lab with a manual flagging system, there is still room to improve by implementing an automatic flagging system.</jats:p> </jats:sec>	[ { "section_content": "Pseudohyperkalemia has been widely reported in acute or chronic lymphocytic leukemia [1,2].In contrast, it is very rare in acute myeloid leukemia (AML) [3][4][5][6].Here I reported the first case of pneumatic tube transport (PTT) causing pseudohyperkalemia in acute myeloid leukemia (AML).Despite the manual flagging system for leukocytosis that initially appeared to work, a persistent investigation revealed that the flagging was not functioning in this patient's care.The process of investigation and our plan to improve the flagging system were described. ", "section_name": "Background", "section_num": null }, { "section_content": "A 55 year-old white man was sent to emergency department (ED) for transfusion on April 3, 2024.He was diagnosed with multiple myeloma in 2016 which, despite multimodality treatment, transformed to myelodysplastic syndrome in 2022, culminating in acute myeloid leukemia (AML) in March 2023.He underwent combination chemotherapy for AML until January 2024.In late March, 2024, a bone marrow biopsy showed worsening AML with 80% myeloblasts by CD34 in the core and 70% blasts in peripheral blood.Hydroxyurea was started, but he tentatively chose not to be admitted for further chemotherapy.On April 2, 2024, he received potassium chloride 40 mEq intravenously (i.v.) as an outpatient for low plasma potassium (K + ) at 2.8 mEq/L.He had been on fludrocortisone and hydrocortisone for adrenal insufficiency that developed in 2019 and also on oral potassium chloride (20 mEq p.o. bid) for chronic hypokalemia that developed in May 2023. Then on April 3, 2024, a blood test (Sample A, Table 1), drawn at his home by a home health nurse and sent to our hospital lab via courier service and walk, showed a hemoglobin of 4.5 g/dL (compared with 7.5 g/dL two days prior) and K + of 3.0 mEq/L.Immediately he was referred to our ED for transfusion.Exam revealed normal vital signs without acute distress. In ED, blood test showed white blood cells (WBC) of 206.8 × 10 3 /µL with 90% blasts, hemoglobin of 7.9 g/ dL and platelets of 38 × 10 3 /µL.Obviously hemoglobin of 4.5 g/dL was a lab error.His Met 10 panel (Sample B, Table 1) showed K + 6.7 mEq/L.The ED physician requested a redraw of blood (Sample C, Table 1) revealing K + 7.8 mEq/L.Hemolysis was ruled out by observing the supernatant of centrifuged blood after the chemistry blood test was completed.Other blood tests included phosphorus 2.6 mg/dL, ionized calcium 5.0 mg/dL, uric acid 4.7 mg/dL, glucose 193 mg/dL, BUN 9 mg/dL and creatinine 1.2 mg/dL.Despite that he had no associated symptoms and his EKG showed no hyperkalemic changes, anti-hyperkalemic treatment with calcium gluconate (i.v.), sodium bicarbonate (i.v.), regular insulin with 50% dextrose (i.v.) and albuterol nebulizer, was immediately administered.Nephrology consult was called for possible need of hemodialysis.The nephrologist on-call rightly suspected pseudohyperkalemia and recommended obtaining a heparinized plasma sample to re-test.The ED physician relayed the request to the Chemistry lab team who addressed the issue appropriately, and a repeat blood K + came back at 3.6 mEq/L (Sample D, Table 1).Pseudohyperkalemia was thus ascertained, although the recommendation of obtaining heparinized plasma was irrelevant because his blood was already heparinized (Table 1).Of note, Sample D was obtained about 30 min after started on anti-hyperkalemic treatment.After the K + was shown to be normal, one unit of packed red blood cells was transfused.Choosing comfort care, he was discharged home with stable vital signs the next day (April 4, 2024) and died one week later. ", "section_name": "Case presentation", "section_num": null }, { "section_content": "The next day (April 4, 2024), I took over the nephrology service and started to investigate the process that had led to the correct diagnosis, with a goal of preventing similar incidents.When I entered the main lab of the 1).But I still wondered why there was a redraw (Sample C) if the flagging system was working.The high K + from Sample B together with the flagging of leukocytosis from Hematology LT/CLS should have made Chemistry LT/ CLS take appropriate actions immediately.Then pseudohyperkalemia would be readily identified, obviating the need of therapy and nephrology consultation.One month later I went to the lab again, and two other lab LT/CLS gave me the same answer as before.A few days later, I asked the lab supervisor to talk to the LT/CLS who worked that night.Three days later, he told me that the Hematology LT/CLS did not inform Chemistry LT/ CLS, although all lab staff (including Hematology team and Chemistry team) worked in the same large room.Only after clinicians' reminder of possible pseudohyperkalemia did the Chemistry LT/CLS take the appropriate steps, solving the problem.The main finding was that our manual flagging system requires Hematology LT/CLS to make Chemistry LT/CLS and phlebotomists aware of potential issue with elevated WBC counts.We also found that only a Technical Specialist (a very senior CLS who has worked as CLS for more than 15 years) in Chemistry can add the flagging comment in middleware (which is viewable only to lab staff ).Technical Specialists are not on shift 24/7 to add the comment, creating another opportunity to miss these specimens.After this incident, our laboratory is investigating the options of using the Laboratory Information System to automatically flag the results and CLS to make the chemistry team more aware of potentially erroneous K + results due to pseudohyperkalemia. ", "section_name": "Investigation", "section_num": null }, { "section_content": "Since spurious hyperkalemia was first described by Hartmann and Mellinkoff in 1955, there have been abundant case reports of pseudohyperkalemia (falsely elevated potassium in serum) and reverse pseudohyperkalemia (falsely elevated potassium in plasma) [1,2].Avelar compiled a relatively comprehensive list of causes for pseudohyperkalemia [7].All of these same factors such as mechanical stress also apply to reverse pseudohyperkalemia [2,7].Pseudohyperkalemia is mostly associated with severe thrombocytosis or leukocytosis as in acute or chronic lymphocytic leukemia, with one of the main mechanisms being in vitro potassium release into serum from cells during clotting formation [1,2].Another theory suggests that severe leukocytosis has higher consumption of metabolic fuels that may lead to impaired Na + /K + ATPase pump activity, which leads to potassium release from the high number of WBCs [2].Heparinmediated cell membrane damage during processing and centrifugation in the context of hematologic malignancy is a risk for reverse pseudohyperkalemia [7].In contrast, the formation of a fibrin clot in serum specimens is hypothesized to entrap and stabilize tumor cells during centrifugation, avoiding pseudohyperkalemia [8]. Pneumatic tube transport (PTT) system was famously introduced to Mayo Clinic by Dr. Henry Plummer in 1928 and now is being used by many hospitals around the world [9].For example, our hospital's PTT, delivering about 4,000 packages a day, can reach a linear speed of 17 mph with rapid accelerations and decelerations, as well as angular velocity changes with turns.Pneumatic transport of specimens with normal WBC counts did not affect the potassium measurement [10,11].However, Kellerman, et al., described the first case of pseudohyperkalemia caused by pneumatic tube transport of blood specimens from a patient with extreme leukocytosis [11].The patient apparently had acute lymphocytic leukemia transformation originating from his prior mantle-cell non-Hodgkin lymphoma.The different effect of PTT on specimen with normal WBC counts vs. extreme leukocytosis was attributed to both high WBC number and cellular fragility of malignant cells.His study effectively excluded other factors such as vacutainer draw from a venous catheter, venous sample draw, lithium heparin (vs.heparin alone) as a cause of pseudohyperkalemia.In essence, Kellerman's and our case were both reverse pseudohyperkalemia, although no serum sample was tested. In a striking comparison to lymphocytic leukemia (acute or chronic), AML has been rarely associated with pseudohyperkalemia.This may be due to different cellular fragility between lymphocytic leukemia cells and AML blasts.There are only four reported cases, published in English, on pseudohyperkalemia related to AML (3-6 see Table 2).One of the cases was published in French with an English abstract.An attempt to reach the author by email for case details was unsuccessful [5].The falsely elevated serum potassium level in all of the other 3 cases was due to blood clotting.Our patient was the first case of PTT causing pseudohyperkalemia in AML.The reduction of K + by the medical treatment was expected to be at most 1.80 mEq/L in our patient (0.90 mEq/L each for insulin and albuterol, respectively, while no change was expected with sodium bicarbonate) [12].Thus the plasma K + would, at best, be lowered from 7.8 to 6.0 mEq/L which was still much higher than 3.6 mEq/L (the real K + level of our patient).Therefore there was certainly a significant component of pseudohypokalemia.Clotting did not play a role because all blood samples were heparinized. Prompt diagnosis of pseudohyperkalemia is critical to avoid iatrogenic hypokalemia from treatment (including emergency hemodialysis) for pseudohyperkalemia [5][6][7]11].When significant leukocytosis, thrombocytosis, hyperproteinemia, or hyperlipidemia are present, whole blood potassium measurements should be utilized [13].In emergent scenarios where potassium levels are critically needed for subsequent decision-making, whole blood testing should also be utilized [13].Our case indicates that even with lab's manual flagging system for leukocytosis, pseudohyperkalemia can still occur.This can suddenly happen to the same patient in the same lab on the same day.On suspicion of pseudohyperkalemia in the presence of leukocytosis, a whole blood sample should be walked to the lab, without delay, for chemistry test. Through this investigation, the lab realized that our manual flagging is not functioning all the time.The main lesson learned from this incident is to automate the lab flagging system, in an attempt to eliminate pseudohyperkalemia associated with leukocytosis, and in a broader range, to prevent other false lab values.The potential benefit is enormous if all hospitals and labs in the world are considered. In conclusion, pseudohyperkalemia associated with leukocytosis still occurs.This is the first case of pneumatic tube transport causing pseudohyperkalemia associated with AML.When significant leukocytosis, thrombocytosis, hyperproteinemia, or hyperlipidemia is present, whole blood should be utilized for potassium measurements and walked to the lab instead of sent by pneumatic tube transport.Even in a lab with a manual flagging system, there is still room to improve by implementing an automatic flagging system. ", "section_name": "Discussion and conclusions", "section_num": null } ]	[ { "section_content": "none. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "No funding was obtained for this study. ", "section_name": "Funding", "section_num": null }, { "section_content": "No datasets were generated or analysed during the current study. ", "section_name": "Data availability", "section_num": null }, { "section_content": "Acute myeloid leukemia LT/CLS Lab technicians/Clinical lab scientists PTT Pneumatic tube transport Yangming Cao is the sole author of this article and takes full responsibility for its content. Ethics approval and consent to participate Not applicable.This article does not contain any studies with animals performed by the author. Not applicable due to the death of the patient. the author has no conflict of interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Abbreviations", "section_num": null }, { "section_content": "", "section_name": "Abbreviations", "section_num": null }, { "section_content": "Acute myeloid leukemia LT/CLS Lab technicians/Clinical lab scientists PTT Pneumatic tube transport ", "section_name": "AML", "section_num": null }, { "section_content": "Yangming Cao is the sole author of this article and takes full responsibility for its content. ", "section_name": "Author contributions", "section_num": null }, { "section_content": "Ethics approval and consent to participate Not applicable.This article does not contain any studies with animals performed by the author. ", "section_name": "Declarations", "section_num": null }, { "section_content": "Not applicable due to the death of the patient. ", "section_name": "Consent for publication", "section_num": null }, { "section_content": "the author has no conflict of interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's note", "section_num": null } ]
10.4322/acr.2021.278	De novo double-hit B-cell precursor leukemia/lymphoma - an unusual presentation as peritoneal lymphomatosis	Peritoneal lymphomatosis (PL) is a rare presentation of extranodal precursor leukemia/lymphoma.The presentation is often non-specific, leading to delayed diagnosis and treatment.In this case, though the preliminary diagnosis was established on ascitic fluid cytology, the disease progressed rapidly, leading to demise before initiating chemotherapy.Immunophenotyping and molecular studies, performed later, established a diagnosis of de novo B-cell precursor leukemia/ lymphoma with MYC, BCL2 rearrangements (Double-hit lymphoma).MYC, BCL2 rearrangements are rarely reported in precursor B-lymphoma/leukemia which carry dismal prognosis.In this report, we illustrate autopsy findings of PL in an elderly gentleman who presented with ascites for evaluation.	[ { "section_content": "Peritoneal lymphomatosis (PL) is a rare presentation of extranodal Non-Hodgkin lymphoma (NHL).Since the clinical presentation of PL is non-specific, the diagnosis is often delayed.Peritoneal carcinomatosis accompanied by malignant ascites is relatively common; however, 'peritoneal lymphomatosis' is rarely reported in the literature. 1 Differentiation of PL from other morbid entities with similar imaging features such as tuberculous peritonitis, peritoneal carcinomatosis, and peritoneal mesothelioma is difficult without a histological diagnosis. 2In the present genomic era, the appropriate immunohistochemical and molecular workup of lymphoma is indispensable for guiding therapy and prognostic information.The 2016 revised World Health Organization (WHO) classification of lymphoid neoplasms has included the category of high-grade B cell lymphomas (HGBLs) with combined MYC and BCL2 and/or BCL6 rearrangements termed as double-hit (DH) or triple-hit (TH) respectively. 3,4DH is usually reported commonly in diffuse large B-cell lymphoma (DLBCL), less frequently in follicular lymphomas and rarely in B-lymphoblastic lymphoma/ leukemia (B-LBL). 4,5There is limited information on cases of B-LBL with MYC and BCL2 rearrangement.In addition, there are cases of HGBL which can express Tdt rendering a diagnostic dilemma.Herein, we present autopsy findings of an elderly gentleman who presented with ascites for evaluation. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "A 55-year-old gentleman presented with abdominal distension to the gastroenterology service.The distension was insidious in onset, along with decreased urine output over the last 20 days.On examination, the patient had tense ascites with bilateral pitting pedal edema.There was no history of jaundice, hematemesis, melena, altered sensorium, fever, night sweats, or weight loss.Ultrasound revealed ascites with mild hepatomegaly and hydroureteronephrosis of the right kidney.Upper gastrointestinal endoscopy did not reveal any varices.Hepatotropic viruses' markers were negative.His renal function tests were deranged.Blood urea was 128 mg/dl (reference range [RR]; 15-40mg/dl) and serum creatinine was 2.9 mg/dl (RR; 0.6-1.2mg/dl).The liver function test on admission day was as follows: Total bilirubin: 0.4 mg/dl (RR: 0.2 -1.2 mg/ dl), conjugated bilirubin 0.2 mg/dl (RR: 0 -0.3 mg/dl); AST: 37 (RR: 2-40 U/L) ALT: 32 (RR: 2-41 U/L) alkaline phosphatase: 62 IU/L (RR; 44-147 IU/L).However, terminally (3 days after admission), the AST/ALT/ ALP were 408/90/2042 IU, respectively.Abdominal ultrasonography did not reveal features of acute Budd-Chiari syndrome such as thickened walls, intraluminal echogenicity, and compressed hepatic veins or inferior vena cava. The ascitic fluid analysis revealed a high serum ascites albumin gradient (SAAG) ascites (1.2 g/dl).Initial ascitic tap showed sheets of polymorphs, and the subsequent cytology showed numerous intermediate size lymphoid cells with a high nuclear/cytoplasmic ratio.Immunocytochemistry showed lymphoid cells strongly positive for CD45, CD10, Tdt, and C-MYC.Focal positivity for CD20, CD19, CD79a was seen in these atypical lymphoid cells, and a working diagnosis of B-cell lymphoblastic lymphoma/leukemia was kept.Peripheral blood smear did not show blasts.CSF tap did not show atypical cell infiltration.CECT abdomen and PET-CT scan were deferred due to deranged renal function test.Differentials for ascites were considered based on the serum-ascites-albumin gradient (SAAG).Cutoff of >1.1mg/dl is considered as high SAAG, which includes conditions like cirrhosis, heart failure, Budd-Chiari syndrome, sinusoidal obstruction syndrome.Low SAAG includes malignancy, infection, and pancreatitis, among others.The ultrasonography, endoscopy, and hepatic viral markers were negative for cirrhosis, varices, and viral hepatitis, respectively; enabling a rapid ascitic fluid malignant cytology analysis.It is interesting to note that despite being a case of PL, this case showed high SAAG ascites. Since the initial two ascitic fluid taps showed neutrophils, the patient was empirically started on IV cefoperazone-sulbactum, oral acyclovir, oral fluconazole and twice weekly cotrimoxazole.Low molecular heparin was given as deep venous thrombosis (DVT) prophylaxis.Fluconazole and cotrimoxazole were withheld later in view of worsening liver function tests (LFT), and acyclovir was also stopped in view of renal failure.Pre-emptive steroids and chemotherapy could not be started because of the poor general status of the patient.The patient's renal and liver function deteriorated, and he had a sudden cardiac arrest and could not be revived despite resuscitation efforts.A complete autopsy was performed after obtaining informed written consent from the patient's next of kin. ", "section_name": "CASE REPORT", "section_num": null }, { "section_content": "The peritoneal cavity yielded 2 liters of strawcolored fluid.The mesentery and the omentum appeared thick and nodular, forming adhesions to the intestinal wall (Figure 1A).Either side of the diaphragm showed similar greyish white deposits.The liver weighed 2300 g (RR;1330-2100g) and was diffusely enlarged.The porta hepatis showed a greyish white mass measuring 4x3x2cm infiltrating the hepatic parenchyma (Figure 1B).This tumor also infiltrated the pancreas, stomach, the serosal surfaces of the small and large intestine and right kidney.A similar deposit was seen in the periadrenal fat.No significant lymph node enlargement was noted.The heart and brain were grossly and microscopically unremarkable. The microscopy revealed relatively uniformappearing atypical lymphoid cells with blastoid morphology.These cells were infiltrating the peritoneum, porta hepatis, renal pelvis, serosa and mucosa of the gastrointestinal tract (Figure 1C).The cells were intermediate-sized with round to convoluted nuclei with condensed nuclear chromatin, indistinct nucleoli, and scanty cytoplasm.These cells were positive for CD45, CD20, CD10, TdT, CD99 (dotlike), Bcl2 (>70%), and C-MYC (>40%) while they were negative for CD3, CD5, CD34, CD23, Bcl6, Mum1, Cyclin D1 and SOX11 (Figure 1D, 2A, 2B, 2C and 2D). Immunophenotypic diagnosis of B-lymphoblastic lymphoma/leukemia was considered.The fluorescence in-situ hybridization (FISH) was positive for BCL2 rearrangement (18q21) (Vysis LSI BCL2 Dual Color Break Apart Rearrangement Probe) (Figure 3A) and CMYC-IGH fusion (IGH/MYC/CEP 8 Tri-Color Dual Fusion Probe) (Figure 3B).However, FISH was negative for BCL6, BCR-ABL-1, KMT2A translocation, ETV6-RUNX1 translocation, and chromosome-21 amplification.Real-time PCR was negative for CRLF2 overexpression.Copy number abnormalities revealed homozygous deletions of Exon 2 & 4 of CDKN2A and Exon 2 of CDKNA2B (Figure 4).Bone marrow biopsy was normocellular with adequate representation of all marrow elements.There was no infiltration by lymphoma or involvement by leukemic process.The peripancreatic and peribiliary lymph nodes did not show the lymphomatous process.The autopsy did not reveal features of hepatic venous outflow obstruction.There was no evidence of pulmonary thromboembolism.There was focal subpleural infiltration through the diaphragm (direct extension) by lymphoma on the right side. ", "section_name": "AUTOPSY FINDINGS", "section_num": null }, { "section_content": "Non-Hodgkin lymphoma presents primarily as an extranodal disease in 25-40% of cases. 6It can affect organs and sites such as the biliary tree, liver, spleen, gastrointestinal tract, adrenals, peritoneal cavity, among others.Peritoneal surfaces can be involved by malignancies from all three cell-lineages: epithelial (carcinomatosis), mesenchymal (sarcomatosis), and lymphoid (lymphomatosis).Peritoneal lymphomatosis (PL) is defined as the \"intraperitoneal spread of lymphoma\".It can be seen either in association with a primary visceral site of involvement, or without any visceral involvement. 7,8The route of dissemination is postulated to be through visceral peritoneal surfaces, gastrocolic ligament, and transverse mesocolon.PL is an exceptional clinical scenario, which is frequently associated with primary gastrointestinal NHL (high grade) and is radiologically identical to peritoneal carcinomatosis.Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma presenting as PL.Multiple patterns of PL include infiltrative mass, distinct nodular, and ascites. 8In the present case, there was a diffusely infiltrative mass presenting with ascites. Based on the immunohistochemical and molecular workup, the present case was a de novo B-LBL with MYC, BCL2 rearrangements.Double-hit/triplehit lymphoma is characterized by MYC (8q24) rearrangement in combination with a BCL2 (18q21) and/ or a BCL6 (3q27) rearrangement. 9,10Double-hit lymphomas (DHL) are rare and represent 4-8% of all diffuse large B-cell lymphomas.They tend to occur in extranodal sites.Four morphological variants have been described in DHL; DLBCL-like, features previously designated as B-cell lymphoma unclassifiable (BCL-U) in 2008 WHO classification, Burkitt lymphoma-like and lymphoblastic lymphoma-like . 4,11ere are few case reports of B-ALL with double hit genetics reported in literature. 12,13In most of those cases, the blasts were L3 type (French-American-British classification) and majority of them had acute leukemia like presentation.Few of the cases had a leukemic transformation from DLBCL.Conversely, on the other end of the spectrum, there can be acute lymphoblastic leukemia-like high grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement lacking Tdt and other immaturity markers. 14Ok et al. 15 described a series of 13 cases of high-grade B-cell lymphomas with TdT expression grouped into three categories.These included cases of de novo high-grade B-cell lymphoma with Tdt expression as well as blastic transformation of a low grade B-cell lymphoma which acquired Tdt during relapse.Features that favoured HGBL over LBL were positivity for BCL6 and monotypic surface immunoglobulin.All the cases showed dismal outcome, despite appropriate therapy. In the present case, copy number analysis revealed homozygous deletions of Exon 2 & 4 of CDKN2A and Exon 2 of CDKNA2B gene.These genes are involved in cell cycle regulation and homozygous deletion carries poor prognosis in ALL. 16ere are two reported cases of doublehit lymphoma expressing Tdt and presenting as ascites. 17,18Unlike ours, one case was a transformation from follicular lymphoma, and the other had involvement of the pancreas primarily.Primary effusion lymphoma (PEL) driven by HHV-8 is another differential.PEL cells typically display a \"null\" lymphocyte phenotype, i.e., CD45 is expressed, but routine B-cell (including surface and cytoplasmic immunoglobulin, CD19, CD20, CD79a) and T-cell (CD3, CD4, CD8) markers are absent.Instead, various markers of lymphocyte activation (CD30, CD38, CD71, human leukocyte antigen DR) and plasma cell differentiation (CD138) are usually displayed. 19Peritoneal lymphomatosis is treated non-surgically and often shows dramatic improvement with chemotherapy.Therefore, early and precise diagnosis is of utmost importance.However, PL with double-hit phenotype has a belligerent course and a poor outcome despite appropriate treatment. 20uble-hit genetics (MYC, BCL2 rearrangement) have inferior outcome with standard chemotherapy.This case highlights that double hit genetics can be seen in B-precursor lymphoma/leukemia and a separate classification of this entity may be beneficial for prognostication and may facilitate further research and design the novel therapeutic approaches such as MYC and BCL2 inhibitors. ", "section_name": "DISCUSSION", "section_num": null } ]	[ { "section_content": "Autops Case Rep (São Paulo).2021;11:e2021278 This work was carried out at the Post Graduate Institute of Medical Education and Research (PGIMER).Chandigarh, India. ", "section_name": "", "section_num": "" }, { "section_content": "• There should be early institution of empirical treatment while awaiting diagnostic results as it has an aggressive course and poor outcome; • De novo double-hit B-cell precursor leukemia/lymphoma needs separate recognition for facilitating further research and targeted therapy. Authors' contributions: Balamurugan Thirunavukkarasu was involved in data collection, literature review and drafting of the manuscript.Amanjit Bal contributed to drafting of the manuscript and supervised it.Balamurugan Thirunavukkarasu, Amanjit Bal and Prateek Bhatia were involved in the diagnosis on autopsy.Jayanta Samanta managed the patient clinically.All authors have read and approved the manuscript. Ethics statement: Informed consent by the next of kin was retained by the institution where the autopsy was performed. Conflict of interest: None. Financial support: None. ", "section_name": "CONCLUSIONS", "section_num": null } ]
10.5334/jcr.147	Differential Expression of Circadian Genes in Leukemia and a Possible Role for Sirt1 in Restoring the Circadian Clock in Chronic Myeloid Leukemia	Disregulation of genes making up the mammalian circadian clock has been associated with different forms of cancer. This study aimed to address how the circadian clock genes behave over the course of treatment for both the acute and chronic forms of leukemia and whether any could be used as potential biomarkers as a read-out for therapeutic efficacy. Expression profiling for both core and ancillary clock genes revealed that the majority of clock genes are down-regulated in acute myeloid leukemia patients, except for Cry2, which is up-regulated towards the end of treatment. A similar process was seen in acute lymphocytic leukemia patients; however, here, Cry2 expression came back up towards control levels upon treatment completion. In addition, all of the core clock genes were down-regulated in both chronic forms of leukemia (chronic myeloid leukemia and chronic lymphocytic leukemia), except for Cry2, which was not affected when the disease was diagnosed. Furthermore, the NAD(+) - dependent protein deacetylase Sirt1 has been proposed to have a dual role in both control of circadian clock circuitry and promotion of cell survival by inhibiting apoptotic pathways in cancer. We used a pharmacological-based approach to see whether Sirt1 played a role in regulating the circadian clock circuitry in both acute and chronic forms of leukemia. Our results suggest that interfering with Sirt1 leads to a partial restoration of BMAL1 oscillation in chronic myeloid leukemia patient samples. Furthermore, interfering with Sirt1 activity led to both the induction and repression of circadian clock genes in both acute and chronic forms of leukemia, which makes it a potential therapeutic target to either augment existing therapies for chronic leukemia or to act as a means of facilitating chronotherapy in order to maximize both the effectiveness of existing therapies and to minimize therapy-associated toxicity.	[ { "section_content": "Important mammalian physiological processes such as sleep-wake patterns, gene transcription and regulation of metabolism to name a few; undergo periodic changes or cir-cadian rhythms which typically span a 24h cycle [1][2][3].Two components underpin the circadian system in mammals: A central pacemaker resident within the suprachiasmatic nucleus (SCN) found in the anterior hypothalamus together with core circadian genes that collectively make up the circadian oscillator and secondly peripheral oscillators located in different tissues around the body [3,4].In order for an organism to respond appropriately to light and dark cycles, a fully functional circadian clock is of paramount importance.Circadian oscillators make use of transcriptional-translational feedback loops that rely on both positive and negative cues from oscillators which in turn causes the characteristic daily cycling of both clock genes as well as clock controlled genes [5] (Yang and Sheng-Fun, 2016).In addition, epigenetics also plays a critical role in regulating the daily periodicity of the circadian clock [6]. Disruption in circadian rhythms appears to be a hallmark for a number of diseases including diabetes and cancer [7].There is an accumulating body of evidence showing a link between disruption of the circadian clock and pathogenesis of cancer.Per1 and Per2 are downregulated in both sporadic and familial forms of breast cancer when compared to normal breast tissue [8].In leukemia, a progressive, malignant disorder that affects the bone marrow and other blood-forming organs resulting in the abnormal production of leukocytes, changes in circadian genes have also been reported.For example, epigenetic changes through the hyper-methylation of the BMAL1 promoter has been associated with the development of hematological malignancies such as non-Hodgkin lymphoma and acute lymphocytic leukemia (ALL), and hyper-methylation of the Per3 gene is manifested in chronic myeloid leukemia (CML) in peripheral blood [9].A comprehensive understanding of how circadian genes behave in leukemia will not only provide insights into how the molecular clock is impacted in this disease but such an understanding could also be used to develop treatment strategies whereby drugs are administered to patients in leukemia in synchrony with their molecular clocks to facilitate greater effectiveness of drug administration to the patients (a process known as chronotherapy). Although altered expression of circadian genes has been previously reported in chronic and acute leukemia [9][10][11], no study has looked at how circadian genes change over the course of treatment for leukemia and during disease relapse in the case of AML and upon diagnosis and during the course of treatment for chronic lymphocytic leukemia (CLL) and whether these genes could be used as biomarkers to assess therapeutic efficacy.Furthermore, we currently lack an understanding of how the oscillation of clock genes is altered in both acute and chronic forms of leukemia and what role if any sirtuin (Sirt1) has in regulating the circadian clock in leukemia given that Sirt1, an NAD(+) -dependent protein deacetylase, has previously been shown to regulate circadian clock gene expression in other cellular systems by directly binding to CLOCK-BMAL1, which in turn promotes both the de-acetylation and degradation of Per2 [12]. In the present study we first investigated the changes in circadian clock gene expression across different forms of leukemia at the point of disease diagnosis, upon treatment completion and in the case of AML during disease relapse with a view to identifying circadian genes as potential biomarkers that could be used to assess treatment efficacy.Secondly, the oscillation of core and ancillary circadian genes were analyzed in both acute and chronic forms of leukemia.Finally, we probed the role of Sirt1 in regulating circadian gene expression in vitro using a small molecule approach in primary cells derived from patients representing both acute and chronic forms of leukemia to see whether Sirt1 it has any role in regulating the circadian clock in both acute and chronic forms leukemia. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "Peripheral blood samples were collected from patients and healthy individuals at the Ministry of National Guard Health Affairs (MNGHA), King Abdulaziz Medical City, Riyadh following signed consent after IRB approval and according to guidelines established at MNGHA.In total the following numbers of patients were recruited for the study, which was spread across different stages of the disease and treatment: 26 patients with acute myeloid leukemia (AML), 22 patients with acute lymphoid leukemia (ALL), 13 patients with chronic myeloid leukemia (CML) and 14 patients with chronic lymphoid leukemia (CLL).In addition, 30 healthy donors (controls) were also recruited for comparative data analysis purposes.Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Leucosep tubes (Greiner Bio-One) according to a previously described method [13].The blood withdrawal from patients and PBMCs preparation were performed between 8 and 10 o'clock in the morning, as all the patients came to the clinic in the morning and also for us to avoid a time shift issue between samples. ", "section_name": "Patients, healthy individuals and samples", "section_num": null }, { "section_content": "PBMCs used in this study were maintained and propagated in DMEM media (UCF) supplemented with 2 mM glutamine, 1X non-essential amino acids, 100 U/ml penicillin, 100 U/ml streptomycin and 10% fetal bovine serum (ATCC).All cells were incubated in a humidified atmosphere of 5% CO 2 and 95% air at a constant temperature of 37°C unless otherwise specified.The Sirt1 inhibitor used in this study, EX527, was purchased from TOCRIS and was received in lyophilized form.100 mM stocks of the inhibitor were prepared using dimethyl sulphoxide (DMSO).Working concentration of EX527 (30 mM) was added to cells directly in culture media.All compound treatments were performed in 6, 12 or 24-well plates (Corning). ", "section_name": "Cell culture and compound treatment", "section_num": null }, { "section_content": "Total RNA from each sample was extracted using the Pure-Link RNA mini kit (ThermoFisher Scientific) according to the manufacturer's instructions.Following extraction, the quantity and quality was assessed using the Nanodrop 8000 8-well spectrophotometer (ThermoFisher).Firststrand synthesis was performed on 400 ng of total RNA using the high capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturers recommendation using the following PCR cycling parameters: 25°C for 10mins, 37°C for 120mins, 85°C for 5mins and finally held at 4°C. ", "section_name": "RNA isolation and synthesis of cDNA", "section_num": null }, { "section_content": "The expression of seven circadian clock genes Per2, BMAL1, Cry1, Cry2, Clock, REV-ERBa and PPARa as well as two circadian-modifier genes, Sirt1 and c-myc, were analyzed using Taqman expression assays (Table 1).Glyceraldehye-3-phosphate dehydrogenase (GAPDH) expression was used as the internal control.All reactions were performed in a MicroAmp optical 384-well plate (Applied Biosystems) in a final volume of 5 ul per reaction.For each reaction, 1 ul of CDNA was used as the input for amplification. Amplification was performed using the 7900 HT Fast Real-time PCR system (Thermofisher) and the PCR cycling parameters were as follows: 95°C for 10mins followed by 40 cycles of PCR reactions at 95°C for 30 sec and 60°C for 1 min.The relative expression levels of the genes assayed in the cell synchronization validation method experiment were calculated using the comparative threshold cycle Ct (∆∆Ct) method.Initially, the Ct value of each gene was normalized to the corresponding Ct value of GAPDH for the same sample to obtain the relative threshold cycle (∆Ct).Following this the ∆Ct for each biological replicate was then exponentially transformed into ∆Ct expression by calculating 2 raised to the -∆Ct.Next, the average and standard deviation of the biological replicates were calculated followed by normalization to the average of the control samples for the same gene (∆∆Ct).Finally the ∆∆Ct was calibrated from control samples and expressed as a relative fold-change.For relative mRNA analysis, raw Ct values were exported from ABI 7900 and imported into Microsoft Excel.Ct values were then used to calculate copy number for each well using historical/generic values from standard curves (Intersection at 0 = 40, slope = -3.5)(Data not shown). Copy numbers were then normalized to GAPDH and mRNA levels were expressed relative to control samples. ", "section_name": "Quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) analysis of mammalian circadian genes", "section_num": null }, { "section_content": "PBMC populations were synchronized using three different methods.One involved culturing cells initially in media containing 10% Fetal Bovine Serum (FBS), another involved culturing cells in 50% FBS for 1hr at 37°C/5% CO 2 followed by culture in 10% FBS, and the third method involved culturing the cells at 32°C for 12 hours followed by 37°C culture for 12 additional hours before use. ", "section_name": "Cell synchronization", "section_num": null }, { "section_content": "Statistical differences between clock gene expression in leukemia patients and control samples were determined using the student's t-test and assuming unequal variances.P-values < 0.05, 0.01 and **0.001 were considered to be statistically significant.Expression in patient samples was expressed relative to control samples (control samples were given an arbitrary value of 1 and patient samples were calibrated to this). ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "The temperature change method was more effective at synchronizing cell populations Different methods exist for the synchronization of cells within a given population.These include culturing cells under serum-starved conditions to enrich for cells in the G 0 phase of the cell cycle [14,15] and lowering the temperature of cell culture to 30-32°C to arrest cells in the G 1 phase of the cell cycle [16].Other approaches for cell synchronization make use of pharmacological agents that induce cell cycle arrest.For example, treatment with mimosine prevents DNA replication by interfering with replication origins and induces G 1 arrest [17,18] or treatment with hydroxyurea induces S-page arrest by inhibition of an enzyme involved in the biosynthesis of deoxyribonucleotides [19].We decided against taking a pharmacological-based approach for cell synchronization given the possibility of this perturbing clock gene expression. For our study we decided to evaluate three different methods for synchronizing PBMC populations.One involved culturing cells in 10% FBS, another involved culturing cells in 50% FBS followed by switching to 10% FBS and the third involved culturing cells at a lower culture temperature of 32°C for 12 hours followed by a return to 37°C before use.Reducing the temperature of cultures has previously been shown in fibroblasts to slow down cell cycle kinetics and thus enables cells to remain within the G 0 phase of the cell cycle [16]. As Figure 1A shows in the average expressing across three different donors, culturing the cells continuously in 10% FBS failed to show any evidence of oscillation of Per2 and BMAL1 suggesting that the cell population were probably in an asynchronous state.Culturing the cells in high FBS (50%) followed by culture in 10% FBS, appeared to show some evidence of oscillation (Figure 1B) which was consistent with what has been previously described in human PBMC samples [20].However, the large variability seen in these samples suggested that the synchronization of the cells was sub-optimal at best.We found that the temperature change method gave oscillation patterns consistent with what has been previously reported.Per2 expression peaked between 6 and 12 hours whereas BMAL1 expression showed a maximum in the opposite direction during the same time period.Furthermore, at 24 hours, the oscillation patterns of the two circadian genes switched over.Based on these findings all subsequent experiments were performed on cell samples following synchronization using the temperature change method. ", "section_name": "Results", "section_num": null }, { "section_content": "PBMC samples were collected from a total of 26 patients spread out across different stages of the disease (9 samples were derived from patients that had been newly diagnosed with AML, 8 were from patients that had completed treatment and 9 were from patients in whom the disease had relapsed.From these samples total RNA was isolated, reverse-transcribed in to cDNA and amplified by quantitative PCR using probes designed to 7 circadian genes (Per2, BMAL1, Cry1, Cry2, Clock, REV-ERBa (Table 1).Our data demonstrated that upon diagnosis of AML, the core circadian clock genes are down regulated with the exception of CRY1 which is not affected in newly diagnosed patients.Bmal1 is the most affected gene followed by Cry2, Clock and then the Per2 gene.In addition the ancillary clock gene, Rev-ERBa (p < 0.05) is significantly downregulated.PPARa is also down regulated in new and end of treatment AML when compared to samples from healthy individuals (Figure 2B).This is in concordance with a previous study that has shown significant down-regulation in core clock genes in newly-diagnosed AML patients [11].Upon completion of treatment, the core clock genes Cry1 (p < 0.01), Cry2 (p < 0.05) and CLOCK (p < 0.05) showed significant down-regulation compared to controls.The same was also observed for the two ancillary clock genes tested; REV-ERBa (p < 0.05) and PPARa (p < 0.05).Furthermore, upon relapse of the disease, some of the core clock genes showed significant down-regulation; Per2 (p In each experiment, RNA samples were harvested from cell pellets from three different donors at 6hr intervals over a period of 42hrs for qRT-PCR analysis BMAL1 and Per2 expression over this period.ΔΔCt analysis was performed in SDS 5.4 (Applied Biosystems).GAPDH was used as a reference control for all analyses.Average expression across the three donor samples for each synchronization method tested is shown in boxes. < 0.05), BMAL1 (p < 0.05), Cry1 (p < 0.001), and Clock (p < 0.05) as well as the ancillary clock gene, REV-ERBa (p < 0.01).Interestingly, Cry2 (p < 0.01) showed significant upregulation in disease relapse patients compared to control samples.The significant down-regulation of core and ancillary clock genes seen in AML patients at the end of treatment gives rise to the possibility of these genes being used as potential biomarkers to assess efficacy in the treatment of AML and the up-regulation of Cry2 could be used as a potential biomarker to assess relapse status in AML before other diagnostic tests are carried out to confirm that the disease has relapsed in patients. ", "section_name": "Circadian genes show distinct patterns of expression during diagnosis of AML, upon treatment completion, and Cry2 is up-regulated during disease relapse", "section_num": null }, { "section_content": "We next wanted to see whether the observations in circadian gene expression seen in AML patients were also reflected in patients diagnosed with ALL to determine whether similarities existed between the acute forms of both myeloid and lymphoid leukemia.For this reason, 22 patient samples (13 patients newly diagnosed with ALL and 9 patients at the end of treatment) were expression profiled by qRT-PCR for the circadian clock genes.Here we observed complete down regulation of all the core clock genes tested: Per2 (p < 0.05), BMAL1 (p < 0.01), Cry1 (p < 0.05), Cry2 (P < 0.05) and CLOCK (p < 0.01) (Figure 2C) as well as the two ancillary clock genes tested: Rev-ERBa (p < 0.01) and PPARa (p < 0.05) (Figure 2D).Upon completion of treatment none of the core clock genes gave significant differences in expression when compared to the expression in newly diagnosed ALL patients.However, significant differences at the completion of treatment were seen when compared to control patient samples: Per2 (p < 0.05), BMAL1 (p < 0.01), Cry1 (p < 0.05) and Cry2 (p < 0.01).A similar observation was also observed for Rev-ERBa (p < 0.01).Interestingly, the only core clock gene that showed significant changes in expression in patient samples following treatment completion was CLOCK.Levels of CLOCK were observed to have returned to levels seen in healthy patients (Figure 2C).This observation with CLOCK at the end of treatment raises two possibilities.One possibility is that CLOCK expression in ALL patients is more sensitive to therapy or the other possibility is that CLOCK expression returning to pre-disease levels could be a potential biomarker for either treatment efficacy or as a read-out for normal circadian activity in ALL patients following treatment. ", "section_name": "Expression of the CLOCK gene is restored to control levels in ALL patients after treatment", "section_num": null }, { "section_content": "Having analyzed changes in circadian gene expression in both acute forms of leukemia, we next wanted to see how circadian gene expression in chronic forms of leukemia (CML and CLL) contrasted with acute forms of the disease.Initially, we analyzed circadian gene expression in CML patients.For this particular study, 13 CML patients were recruited of which 6 were newly diagnosed with the disease and 7 were patients that had undergone a standard 3-month chemotherapy protocol for CML.Of the core clock genes tested, Per2 (p < 0.05), BMAL1 (p < 0.001), Cry1 (p < 0.001) and CLOCK (p < 0.001) showed significant down-regulation in expression when compared to expres-sion levels in healthy controls.In addition, the ancillary circadian gene Rev-ERBa (p < 0.001) also showed significant down-regulation in patients newly diagnosed with CML (Figure 3A).The only core circadian gene which did not show significant changes in expression in newly diagnosed patients compared to control samples was Cry2. ", "section_name": "Core and ancillary circadian genes are down-regulated in CML patients upon disease diagnosis", "section_num": null }, { "section_content": "Upon the completion of a standard 3-month course of chemotherapy, most of the circadian genes tested showed some increase in expression compared with expression in newly diagnosed patients with the exception being Per2 which did not show any difference (Figure 3A).The interesting observation we saw here was that Cry2 expression in patients that had completed the standard 3-month course of chemotherapy was above levels seen in samples derived from healthy individuals (p < 0.05).This observation could mean one of two things.Either, Cry2 expression was affected by the chemotherapy or Cry2 up-regulation could be a poten- tial marker for treatment efficacy and may be a read-out for the circadian clock returning to normal after treatment. ", "section_name": "Cry2 is up-regulated above control levels following an initial 3-month course of chemotherapy", "section_num": null }, { "section_content": "In contrast to differential circadian gene expression which was observed in CML patients upon diagnosis and after a first course of treatment, the same was not observed in CLL patients.Only a few of the core clock genes showed statistically significant changes in expression upon disease diagnosis: Per2 (p < 0.05), BMAL1 (p < 0.05) and Cry2 (p < 0.05).For the ancillary clock genes tested, only REV-ERBa showed statistically significant differences upon disease diagnosis (p < 0.01) and upon completion of treatment (p < 0.05).These observations do partially concur with what has been previously reported in CLL patients for Per2 and BMAL1 [21] with the exception being the CLOCK gene.However, our observations for Cry2 in CLL patient samples have not been previously reported. ", "section_name": "The majority of circadian clock genes are down-regulated in CLL patients except Cry2 and PPARa expression which are not affected by disease diagnosis or treatment", "section_num": null }, { "section_content": "Although circadian genes have previously been shown to oscillate in human PBMC cells [22], no study has previously been reported which has looked at the oscillation of core and ancillary clock genes in both acute and chronic forms of leukemia.To study the oscillation we derived PBMCs from patients diagnosed with one of the four categories of leukemia (n = 5 for each category of leukemia).Samples for each category were then pooled and synchronized using the temperature change method.Samples were collected every 6h after synchronization and clock gene expression determined by qRT-PCR.Circadian gene oscillations were then analyzed under the following categories (Figure 4): Core (Per2 vs. BMAL1, Cry2 vs. Clock), Ancillary (Rev-ERBa vs. PPARa) and modifier (SIRT1) and c-myc).The expression of c-myc was measured given that it is a direct downstream target of the SIRT1 transcription regulated complex.As expected, control PBMC samples demonstrated circadian gene oscillation over the 30h analysis period. In addition, both core and ancillary clock genes, Sirt1 and c-myc showed oscillation in AML and ALL patient samples.In CLL patient samples, oscillation of BMAL1, Cry2 and CLOCK were observed (Figure 4).However, oscillation was absent in CML patient samples and this absence was present across all the core and ancillary circadian genes tested as well as the modifier genes Sirt1 and c-myc.Our results suggest that in AML, ALL and CLL patients, evidence of a functional clock are present and that in the case of CML patients, the lack of circadian gene oscillations is representative of a dysfunctional circadian clock. Inhibition of Sirt1 using the selective inhibitor EX527 results in some recovery in BMAL1 oscillation in CML patient samples following cell synchronization. Sirt1 is known to regulate genes associated with the circadian clock.As well as being expressed in a circadian manner, a relationship between Sirt1 expression and the acetylation status of BMAL1 and Histone H3 has previously been reported [23].Separately, Sirt1 has also been shown to bind to CLOCK to promote the de-acetylation of BMAL1 at the CLOCK: BMAL1 chromatin complex [23] (Nakahata Y et al., 2008).We wanted to see whether interfering with Sirt1 activity alters the oscillation of circadian genes in leukemia.Following cell synchronization, samples were treated with the Sirt1-selective inhibitor EX527 at 30 uM, a concentration within the range of concentrations known to selectively inhibit Sirt1 in blood cells [24].At this concentration, we saw around 50% inhibition of Sirt1 in PBMC samples derived from healthy patients within 6h of treatment as seen by measuring levels of Sirt1 transcript (Figure 6A).In AML patient samples we saw that inhibition of Sirt1 resulted in \"fine-tuning\" of BMAL1 and Rev-ERBa oscillation.A similar observation was seen in CLL patient samples, where treatment with EX527 resulted in a regularizing of BMAL1 oscillation (Figure 5).The most interesting observation we saw was in CML patient samples, where inhibition of Sirt1 resulted in a return of BMAL1 oscillation which was previously absent in patient samples (Figure 4).Taken together our findings suggest a link between Sirt1 and the oscillation of circadian genes in leukemia. ", "section_name": "Evidence of circadian gene oscillation is present in AML, ALL and CLL patients but is absent in CML patients following cell synchronization", "section_num": null }, { "section_content": "Having observed that inhibition of Sirt1 played a role in the \"fine-tuning\" of circadian gene oscillation and in the case of CML led to a complete recovery of BMAL1 oscillation, we next wanted to see whether treatment of patient samples with EX527 altered the relative expression of the circadian genes and how soon after treatment were these changes noticeable.We chose to analyze both 6h and 18h after treatment.Analysis at 6h after treatment would allow us to identify changes in the early-response circadian genes and analysis at 18h after treatment would allow us to identify changes in late-response circadian genes.In AML patient samples we saw an up-regulation of Cry2 and PPARa and a down-regulation of CLOCK (Figure 6B) at 6h following treatment with EX527.In ALL patient samples, similar to what was observed in AML samples, Cry2 was also up-regulated.However, in ALL patient samples all of the other circadian genes tested were all down-regulated within 6h of treatment with EX527 when compared to non-treated patient samples (Figure 6B).In comparison, the response of the circadian genes to 6h of treatment with EX527 in CML and CLL patient samples was virtually identical (Figure 6B) with the exception of Cry2 which was slightly up-regulated in CML.In contrast all of the circadian genes in all patient categories showed down-regulation (apart from Cry2 in ALL patient samples) at 18h following treatment with EX527 when compared to nontreated samples.We also looked at the relative expression of both Sirt1 and c-myc following treatment of patient samples with the inhibitor EX527.In AML, CML and CLL patient samples at 6h following treatment we saw an up-regulation of sirt1 and c-myc expression compared to non-treated samples (Figure 6D).By 18h post-treatment, sirt1 and c-myc expression was inhibited in AML and ALL samples (Figure 6E).Taking together, our data suggests that inhibition of Sirt1 is a feasible approach to modulating circadian gene activity in leukemic patients as part of a chronotherapy-based approach to treatment of acute forms of leukemia. ", "section_name": "In vitro inhibition of Sirt1 significantly modulated circadian clock gene expression in both AML and ALL compared to CML and CLL patients", "section_num": null }, { "section_content": "In this study we have shown that circadian gene expression is affected in different ways depending on the subtype of leukemia that has been diagnosed and that expression of some of these genes could be used as potential biomarkers to assess therapeutic efficacy.Further we also showed that inhibition of Sirt1 using EX527 may have a role in normalizing BMAL1 oscillation in CML patients.Therefore our work supports the idea of circadian gene expression being altered in different ways depending on the type of leukemia that has been diagnosed and further strengthens the link between leukemia and a disrupted circadian clock. Although circadian gene expression is known to be disregulated in leukemia, no study has thus far looked at how circadian gene expression changes over the course of leukemia, after treatment and in the case of AML during disease relapse.A recent study has shown that disruption in components of the canonical circadian pathway leads to anti-leukemic effects by depleting leukemia stem cells (LSCs) and impairing proliferation and myeloid differentiation [25].Here genetic ablation of BMAL1 in mice resulted in impairment in AML maintenance while sparing normal hematopoiesis.In our study, we saw a downregulation in BMAL1 expression in newly diagnosed AML patients and expression of this gene moved in the direction of control levels in patients who had undergone a relapse of the disease (Figure 2B).Our findings suggests that BMAL1 may play a role during disease by enhancing proliferation and differentiation of LSCs and thus contributing to a more aggressive form of disease which is usually the case during relapse in AML.It would be interesting to isolate LSCs from both AML patients and patients who had undergone a relapse of the disease and compare circadian gene expression and correlate this to LSC proliferation and differentiation studies as this would validate the importance of the circadian clock to LSC biology during relapse of AML. Changes in circadian gene expression, in particular CLOCK have been previously reported in AML, ALL and CML patients when compared to healthy individuals [9][10][11].While our findings in AML do concur with these findings, we observed significant down-regulation of the CLOCK gene in patients newly diagnosed with either ALL (Figure 2C) or CML (Figure 3A).The authors of these particular studies sampled patients indigenous to the Far East, whereas the patients recruited for our study were of Middle-eastern heritage in particular those indigenous to Saudi Arabia.It is entirely possible that the differences we saw in our study could be due to genotypic differences between the two study populations which may in turn lead to differences in circadian gene expression in leukemia. Down-regulation of the core circadian genes BMAL1 and Per2 have previously been reported in patients diagnosed with chronic lymphocytic leukemia (CLL) [21].Further, these changes in circadian gene expression became more apparent in the CLL group when the patients were further sub-divided into groups working regular shifts vs those working irregular shifts suggesting that changes in circadian gene expression were exacerbated in CLL patients that did not have a regular work pattern.In addition dis-regulated expression of clock-controlled genes such as c-myc and cyclin D1 were also observed.Collectively these findings suggest that aberrant expression of clock genes together with apoptosis and proliferation-related genes may be contributing to the development and maintenance of CLL.Although in our study we did also see significant down-regulation of BMAL1 and Per2 in patients newly diagnosed with CLL (Figure 3C), we also saw significant down-regulation in the core clock gene, Cry2 as well as the ancillary clock gene Rev-ERBa (Figure 3C and Figure 3D).Our findings suggest that the dis-regulation of additional core and ancillary clock genes may well be contributing to the etiology of CLL.Interestingly, we also observed that expression of Cry2 was comparable between CLL patients newly diagnosed with the disease and healthy non-leukemic patients suggesting that not all genes making up the core circadian clock are dis-regulated at least in the case of CLL. Therapeutic application of circadian rhythms has been used in the area of heart disease to determine time-of-day biomarkers.Classic biomarkers that have been previously used in cardiovascular disease relate to both patient state (e.g.lifestyle factors, age diet, exercise, smoking etc.) and readouts of biological processes (e.g.assessment of transcriptional and/or protein levels) [26][27][28].Although such biomarkers have traditionally been sampled during the day, more relevant biomarkers termed \"chronobiomarkers\" are novel biomarkers that are sampled over the course of 24 h, thus sampling both during the day and night, and therefore takes into account the circadian rhythms associated with normal physiological and molecular processes.Currently no such chronobiomarkers have been reported for acute and chronic forms of leukemia, which could be used to assess either treatment efficacy or the re-establishment of normal circadian activity in patients following treatment. An important observation from our study was how the expression of the Clock gene returned to similar levels as those seen in healthy patients following treatment for ALL (Figure 2C).Although our finding does support the potential use of Clock gene expression as a novel chronobiomarker in ALL, further studies would have to be performed on larger sample sizes as well as to look at the effect if any of chemotherapy on Clock gene expression to rule out the possibility of therapy intervention having a direct effect on the transcription of the Clock gene. Initial therapy for CML usually involves patients being administered tyrosine-kinase inhibitors such as imatinib [29,30] or Dasatinib [31].Failing this, patients are then usually given an allogeneic stem cell transplant [32].Until now the effect of chemotherapy on circadian gene expression has not been extensively studied.Terazono et al 2008 showed that the treatment of NIH3T3 cells with chemotherapy agent 5-FU for 48 h resulted in a significant reduction of mRNA levels of Period1 (Per1) and Period2 (Per2).Moreover, the injection of 5-FU (2 mg/kg/h) to mice attenuated the oscillation in the expressions of Per1 and Per2 in the liver and suprachiasmatic nuclei, the center of the mammalian circadian clock [33].This paper describes for the first time that the chemotherapy agent could have an influence on clock gene expression and oscillation.Microarray analyses on imatininb responders in CML have shown widespread changes in cell proliferation markers [34].Although we cannot completely rule-out the direct effect of imatinib on circadian gene expression, our data does suggest the potential use of circadian markers such as Cry2, Rev-ERBa and PPARa for assessing response to therapy. Disruption in circadian gene oscillations has previously been shown for the clock genes (Per1, Per2, Per3, Cry1, Cry2 and CKIe) in CML patients, and partial recovery of oscillation in these genes was seen in patients that demonstrated both complete cytogenetic response (CCyR) and major molecular response (MMR) following treatment with imatinib [10].Although we did also observe disrupted oscillations in Per2 and Cry2 in patients newly diagnosed with CML, the lack of oscillation was not just restricted to these circadian genes but also included additional core clock genes such as BMAL1 and CLOCK, as well as ancillary clock genes such as Rev-ERBa and PPARa (Figure 4).Our findings suggest that a complete disruption of both core and ancillary clocks is manifested in CML. In addition to looking at circadian gene oscillation in CML, we also looked for oscillation in the other forms of leukemia, AML, ALL and CLL.Interestingly, we did observe some evidence of circadian gene oscillation in AML and ALL patients suggesting that at least in the case of acute leukemia, a functionally intact circadian clock is present.In CLL patients, disruption in oscillation was restricted to Per2, Rev-ERBa and PPARa and oscillation of BMAL1, Cry2 and CLOCK was largely unaffected (Figure 4).Although dysregulation of some circadian genes is known to take place in CLL [21], our findings suggest that the oscillation of other genes remains largely unaffected by the disease. Another important finding from this study was the \"finetuning\" of core and ancillary clock gene oscillation that as observed when Sirt1 activity was inhibited using the selective inhibitor EX527.In the presence of the inhibitor, BMAL1 and PPARa oscillations were found to be fine-tuned in AML and CLL (Figure 5) patient samples when compared to nontreated patient samples (Figure 4).A role for Sirt1 in regulating circadian gene expression has previously been shown.For example, Sirt1 associates with CLOCK-BMAL1 heterodimers to promote the deacetylation and degradation of Per2 (Asher G et al., 2008).Sirt1 has also been shown to control the circadian expression of genes involved in metabolic pathways in the liver [35].Although a role for Sirt1 in regulating the circadian clock in leukemia has yet to be established, a role for Sirt1 in cancer has previously been demonstrated.Sirt1 has been shown to regulate cell proliferation and apoptosis through epigenetic modification of tumour suppressor genes such as p53 [36] or oncogenes such as β-catenin [37].The restoration of BMAL1 oscillation in CML patients following Sirt1 inhibition suggests Sirt1 may be a feasible drug target to restore core clock oscillation in CML patients as part of a chronotherapy strategy for treating CML. In conclusion, circadian clock gene expression is altered in patients presenting both acute and chronic forms of myeloid and lymphoid leukemia when compared to healthy individuals.Interestingly there is difference in the level of clock gene changes between different patient categories, AML, ALL, CML and CLL.This could be due to the nature of the pathways involved in the malignancy that affect the expression of each clock gene separately in different ways.Further, inhibition of Sirt1 causes the \"fine-tuning\" of circadian gene oscillation for some types of leukemia and in CML patient's resulting in the complete recovery of BMAL1 oscillation.Taken together, these data support the notion that SIRT1 has a role in regulating the circadian clock in leukemia and that the targeting of SIRT1 and therefore the molecular clock using chronopharmacological agents such as EX527 could prove to be a novel and effective therapeutic strategy for improving dysfunctional circadian rhythms in leukemia and thus for both improving the effectiveness of existing chemotherapeutic drugs and reducing drugassociated toxicity. ", "section_name": "Discussion", "section_num": null }, { "section_content": "This paper is dedicated to the soul of Dr. Sabhi Rahman (SR) who passed away just after completing the experimental work and writing the paper. ", "section_name": "Dedication", "section_num": null }, { "section_content": "The additional files for this article can be found as follows: • Table 1 ", "section_name": "Additional Files", "section_num": null } ]	[ { "section_content": "This research was supported by a KAIMRC funded grant (RC12/135).We thank all coordinators, doctors for their assistance with arranging for the collection of patient samples from the National Guard Hospital, Riyadh. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "The authors have no competing interests to declare. Conceived and designed the experiments: SR and MB.Performed the experiments: SR, ASAH, HAJ, AM, TT.Analyzed the data: SR, MB.Contributed reagents/materials/analysis tools: SR, ASAH, AN, AM, TT.Wrote the paper: SR, MB. ", "section_name": "Competing Interests", "section_num": null }, { "section_content": "The authors have no competing interests to declare. ", "section_name": "Competing Interests", "section_num": null }, { "section_content": "Conceived and designed the experiments: SR and MB.Performed the experiments: SR, ASAH, HAJ, AM, TT.Analyzed the data: SR, MB.Contributed reagents/materials/analysis tools: SR, ASAH, AN, AM, TT.Wrote the paper: SR, MB. ", "section_name": "Author Contributions", "section_num": null } ]
10.3324/haematol.2019.225078	Unique clinico-biological, genetic and prognostic features of adult early T-cell precursor acute lymphoblastic leukemia	Funding: this project was supported by the Asociación Española Contra el Cáncer (project ref: GC16173697BIGA), PI14/01971 FIS, Instituto Carlos III, CERCA Program/Generalitat de Catalunya, 2014-SGR225 (GRE), Obra Social “La Caixa” . This work was also partially supported by FEDER funds from the ISCIII (PT13/0010/0026, CIBERONC (CB16/12/00284 and CB16/12/00400), Madrid, Spain). P. Barba was supported by the Instituto de Salud Carlos III FIS16/01433 and PERIS 2018-2020 from Generalitat de Catalunya (BDNS357800) grants	[ { "section_content": "GRAALL 2003 and 2005 trials showed that treatment intensification with allogeneic hematopoietic stem cell transplantation (HSCT) in ETP-ALL with early treatment resistance could improve survival. 12ere we retrospectively analyzed the frequency, clinico-biological and prognostic features of adult ETP-ALL vs. other T-ALL cases in a series of 185 adults with T-ALL treated within two consecutive minimal residual diseaseoriented trials by the Programa Español de Tratamientos en Hematología (PETHEMA): ALL-HR-2003 (NCT00853008) and ALL-HR-11 (NCT01540812), the latter still ongoing). All 185 patients were diagnosed with T-ALL using the WHO criteria.The treatment protocol schedules have been described previously elsewhere. 13,14The patients' characteristics at diagnosis and at follow-up are summarized in Table 1.The immunological T-ALL subtype was defined according to the European Group for the Immunological Characterization of Leukemias (EGIL) criteria after centralized review of immunophenotypic reports.In turn, the criteria proposed by Zuurbier et al. 2 were used to define ETP-ALL.The combination of markers used (<5% CD1a, CD8 and CD4; >25% CD34 and/or CD33/CD13), resembles that proposed by Coustan-Smith et al., while dim CD5 expression (<75% positive cells) was not considered.The inclusion of negativity for CD4 instead of dim CD5 expression as an immunophenotypic criterion, allows identification of most ETP-ALL cases identified by gene expression profiling (increased sensitivity of immunophenotyping to detect ETP-ALL cases). 2 Clinical data were obtained in accordance with the principles of the Declaration of Helsinki and Spanish legislation, after written informed consent had been provided by each patient.The study was approved by the Institutional Review Board of the Hospital Germans Trias i Pujol (Badalona, Spain). Comparisons between ETP-ALL and other T-ALL cases were performed with the χ 2 test, Fisher exact test, and the median test, as appropriate.Event-free survival and overall survival curves were plotted using the Kaplan-Meier method and compared by the log-rank test.A secondary haematologica 2020; 105:e294 Cumulative incidence of relapse and non-relapse-related mortality were analyzed as competing events with the Gray test.A Cox proportional hazard regression model was used to identify predictive factors for event-free and overall survival, whereas multivariate analysis for cumulative incidence of relapse was performed with the Fine and Gray model.Statistical significance was set at P-values <0.05.Full immunophenotypic data according to the EGIL criteria were available for 167/185 T-ALL patients; 34/167 (20%) T-ALL cases met criteria for ETP-ALL as defined by both Zuurbier et al. and the WHO. 7This frequency is similar to that observed in previously published cohorts of adults with T-ALL 11,12 in which the phenotypic criteria applied in early pediatric studies to define ETP-ALL were used. 1 Significant differences were observed when comparing ETP-ALL and non-ETP-ALL cases at diagnosis (Table 1).Patients with ETP traits were older [mean ± standard deviation (SD): 39±12 years vs. 33±12 years, P=0.01], more frequently had lymph node enlargement (79% vs. 56%, P=0.02), had lower white blood cell counts (mean±SD: 72.2±155.1x10 9/L vs. 96.6±111.7 x10 9 /L, P=0.004), and showed a slightly different female to male ratio (13/21 vs. 30/103, P=0.06), similarly to what has been shown for the GRAALL cohort. 12In addition, our ETP-ALL cases frequently showed absence of CDKN2A/B (P<0.001) and TCRG (P=0.003)gene deletions.These results are in line with those previously reported in the literature 6,14,16 and support the use of both genetic markers for the diagnosis of ETP-ALL.However, some discordance between immunophenotypic and genetic results was observed, which might be due to the limited sensitivity of current immunophenotypic criteria, highlighting the need for more precise and robust criteria and standardized methods for the diagnosis of ETP-ALL. ", "section_name": "", "section_num": "" }, { "section_content": "No significant differences in complete response, overall survival, event-free survival and cumulative incidence of relapse rates were observed between ETP-ALL patients enrolled in the two treatment protocols (data not shown).This allowed a combined evaluation of the data from both PETHEMA trials.ETP-ALL patients showed significantly poorer response to induction therapy compared to other T-ALL patients (Table 1).Thus, ETP-ALL patients had poorer bone marrow response (<10% blasts) on day +14 compared to other T-ALL cases (18% vs. 63%, P<0.001); half of ETP-ALL cases required second induction treatment (vs.10% of the remaining T-ALL cases, P<0.001) and 23% of them did not attain a complete response (vs.only 6% of other T-ALL cases, P=0.005).Minimal residual disease data were available for 132/167 patients who reached a complete resposne after one or two induction cycles.Two-thirds (65%) of ETP-ALL cases showed minimal residual disease levels ≥0.1% (vs.18% of non-ETP-ALL cases, P<0.001), the percentage increasing to 85% (vs.37%, P<0.001) at a cut-off level of 0.01%.Consequently, as indicated per protocol, more ETP-ALL patients underwent allogeneic HSCT (70% vs. 21%, P<0.001).Despite treatment intensification with allogeneic HSCT, ETP-ALL patients did not attain a significant improvement in the probability of overall surhaematologica 2020; 105:e295 vival (Figure 1A,B).In fact, differences in overall and event-free survival between ETP-ALL and other non-ETP T-ALL patients were mostly due to the greater rate of failure to induction therapy, with a minimal contribution of the higher (non-statistically significant) cumulative incidence of relapse observed among ETP-ALL patients vs. other T-ALL cases (Online Supplementary Figure S1).However, it should be noted that the overall survival rate of adult T-ALL patients from the PETHEMA cohort who underwent allogeneic HSCT was lower than that observed in other studies. 12,13Thus, the improvement of non-transplant-related mortality in transplanted patients might have a positive impact on survival of ETP-ALL patients.Multivariate analyses showed that ETP-ALL was an independent adverse predictor for both overall survival and event-free survival [hazard ratio of 2.14 (95% confidence interval: 1.29-3.53),P=0.003 and 1.91 (1.18-3.11),P=0.009, respectively] together with a high white blood cell count (Online Supplementary Table S1). ", "section_name": "LETTERS TO THE EDITOR", "section_num": null }, { "section_content": "In summary, here we confirmed the previously reported frequency and clinico-biological features of adult ETP-ALL and showed that adult ETP-ALL patients should be considered as a group with a worse prognosis.The poorer outcome of ETP-ALL patients in our study was due to a lower response to induction therapy and not to an increased relapse rate, which highlights the need to investigate the potential utility of alternative induction therapy for these patients.The use of acute myeloid leukemia-oriented chemotherapy, together with intensification of chemotherapy regimens, has been effective in clearing disease in pediatric ETP-ALL cases refractory to initial induction chemotherapy. 17A similar strategy could be applied to adult ETP-ALL patients.Based on these findings, and given the poor response attained in adults with the conventional induction regimen, it makes sense to explore the potential benefit of the FLAG-IDA regimen (idarubicin; fludarabine; ARA-C and granulocyte colonystimulating factor), a more myeloid-oriented chemotherapy schedule, as front-line induction treatment together with allogeneic HSCT after standard consolidation.In addition, new therapies targeting specific genes altered in ETP-ALL patients, such as FLT3 inhibitors, have emerged as novel alternative treatment strategies.However, the potential benefit of this latter treatment is restricted to ETP-ALL patients carrying FLT3 mutations in the tyrosine kinase domain (1/9, ETP-ALL cases in our cohort; data not shown).Similarly, other potential targeted therapies (e.g.Jak2 inhibitors or Bcl-2 antagonists) may also provide a benefit, but require further investigation. ", "section_name": "LETTERS TO THE EDITOR", "section_num": null } ]	[ { "section_content": "Eulàlia Genescà, 1* Mireia Morgades, 2 Pau Montesinos, 3 Pere Barba, 4 Cristina Gil, 5 Ramon Guàrdia, 6 María-José Moreno, 7 Daniel Martínez-Carballeira, 8 Irene García-Cadenas, 9 Susana Vives, 2 Jordi Ribera, 1 José González-Campos, 10 Celia González-Gil, 1 Lurdes Zamora, 2 José-Luís Ramírez, 2 Marina Díaz-Beya, 11 Santiago Mercadal, 12 María-Teresa Artola, 13 Antònia Cladera, 14 Mar Tormo, 15 Arancha Bermúdez, 16 Ferran Vall-Llovera, 17 Pilar Martínez, 18 María-Luz Amigo, 19 Silvia Monsalvo, 20 Andrés Novo, 21 Marta Cervera, 22 Antoni García-Guiñon, 23 Jordi Juncà, 2 Juana Ciudad, 24 Alberto Orfao 24 and Josep-Maria Ribera 1,2 1 Josep Carreras Leukaemia Research Institute, Campus ICO-Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona; 2 Clinical Hematology Department, ICO-Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona; 3 Hospital Universitari i Politècnic La Fe & CIBERONC, Instituto Carlos III, Madrid; 4 Clinical Hematology Service, Hospital Universitari de la Vall d'Hebron, Barcelona; 5 Clinical Hematology Service, Hospital General de Alicante, Alicante; 6 Clinical Hematology Service, Hospital Josep Trueta-ICO, Girona; 7 Clinical Hematology Service, Hospital Vírgen de la Victoria, Málaga; 8 Clinical Hematology Service, Hospital Universitario Central de Asturias, Oviedo; 9 Clinical Hematology Service, Hospital de la Santa Creu i Sant Pau, Barcelona; 10 Clinical Hematology Service, Hospital Vírgen del Rocío, Sevilla; 11 Clinical Hematology Service, Hospital Clínic de Barcelona, Barcelona; 12 Clinical Hematology Service, Hospital Duran i Reynals-ICO, Hospitalet del Llobregat; 13 Clinical Hematology Service, Hospital Universitario de Donostia, Donostia; 14 Clinical Hematology Service, Hospital Son Llàtzer, Palma de Mallorca; 15 Clinical Hematology Service, Hospital Clínico de Valencia, Valencia; 16 Clinical Hematology Service, Hospital Marqués de Valdecilla, Santander; 17 Clinical Hematology Service, Hospital Mútua de Terrassa, Terrassa; 18 Clinical Hematology Service, Hospital 12 de Octubre, Madrid; 19 Clinical Hematology Service, Hospital Morales Meseguer, Murcia; 20 Clinical Hematology Service, Hospital Gregorio Marañón, Madrid; 21 Clinical Hematology Service, Hospital Son Espases, Palma de Mallorca; 22 Clinical Hematology Service, Hospital Joan XXIII, Tarragona; 23 Clinical Hematology Service, Hospital Arnau de Vilanova, Lleida and 24 Centro de Investigación del Cáncer (IBMCC-CSIC/USAL), Hospital Clínico Universitario de Salamanca, Instituto Bio-Sanitario de Salamanca, CIBERONC Salamanca, Spain Acknowledgments: we would like to thank Rosa Coll, Rodrigo Martino, Raimundo García Boyero, Jesús Mª Hernández Rivas, Pilar Herrera, Juan Bergua, Mª Jesús Peñarrubia, Eloy del Potro, Magdalena Sánchez, Beatriz Soria, Aurelio López Martínez, Mª José Sánchez Sánchez, Pilar Bravo, Pilar Vivancos, Alfons Serrano, José Santiago Bermón, Maria Lourdes Amador Barciela and Matxalen Olivares Salaverri for providing retrospective clinical data for this study.Funding: this project was supported by the Asociación Española Contra el Cáncer (project ref: GC16173697BIGA), PI14/01971 FIS, Instituto Carlos III, CERCA Program/Generalitat de Catalunya, 2014-SGR225 (GRE), Obra Social \"La Caixa\" .This work was also partially supported by FEDER funds from the ISCIII (PT13/0010/0026, CIBERONC (CB16/12/00284 and CB16/12/00400), Madrid, Spain).P. Barba was supported by the Instituto de Salud Carlos III FIS16/01433 and PERIS 2018-2020 from Generalitat de Catalunya (BDNS357800) grants.Correspondence: EULALIA GENESCA' [email protected] doi:10.3324/haematol.2019.225078Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org. ", "section_name": "", "section_num": "" } ]
10.18632/oncotarget.9215	Phosphorylation of Notch1 by Pim kinases promotes oncogenic signaling in breast and prostate cancer cells	Tumorigenesis is a multistep process involving co-operation between several deregulated oncoproteins. In this study, we unravel previously unrecognized interactions and crosstalk between Pim kinases and the Notch signaling pathway, with implications for both breast and prostate cancer. We identify Notch1 and Notch3, but not Notch2, as novel Pim substrates and demonstrate that for Notch1, the serine residue 2152 is phosphorylated by all three Pim family kinases. This target site is located in the second nuclear localization sequence (NLS) of the Notch1 intracellular domain (N1ICD), and is shown to be important for both nuclear localization and transcriptional activity of N1ICD. Phosphorylation-dependent stimulation of Notch1 signaling promotes migration of prostate cancer cells, balances glucose metabolism in breast cancer cells, and supports in vivo growth of both types of cancer cells on chick embryo chorioallantoic membranes. Furthermore, Pim-induced growth of orthotopic prostate xenografts in mice is associated with enhanced nuclear Notch1 activity. Finally, simultaneous inhibition of Pim and Notch abrogates the cellular responses more efficiently than individual treatments, opening up new vistas for combinatorial cancer therapy.	[ { "section_content": "Notch signaling is frequently deregulated in aggressive forms of both hematopoietic malignancies and solid tumors [1][2][3][4].High Notch1 levels have been linked to poor prognosis in breast cancer [5], where Notch1 has been shown to induce epithelial-to-mesenchymal transition (EMT) [6,7], upregulate extracellular matrix metalloproteinases [8], and induce a switch to glycolytic metabolism [9], contributing to both tumor initiation and ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "progression.Similarly in prostate cancer, Notch1 supports cancer cell survival and EMT [10].While deregulated Notch signaling is often involved in metastatic growth as well as therapy resistance, there are also contradictory data, suggesting that the oncogenic effects of Notch are highly dependent on the cellular context [11]. Although the molecular components of Notch signaling have been well defined, the mechanisms regulating Notch activity have not yet been fully characterized [12].The Notch intracellular domain (NICD) represents the active moiety of the receptor and is generated via proteolytic cleavage of the full-length transmembrane protein.Diversity in cellular responses is created by the four different Notch family receptors (Notch1-4), their five different ligands (Jagged1, 2; Delta-like1, 3, 4) [1,12], as well as by the DNA-binding transactivator CSL/RBP-Jκ, which exhibits differential binding preferences to the four NICDs [13][14][15].Signaling activity is regulated by the intersection of Notch with other signaling pathways, including BMP/TGF-β, Wnt, PI-3 kinase and the cellular hypoxic response [9,12].Direct fine-tuning at the molecular level is mediated via post-translational modifications of Notch receptors and ligands.The Notch receptors can be modified by ubiquitylation, sumoylation, hydroxylation, acetylation and phosphorylation, all of which can specify the signaling output [12].Even though Notch family members are phosphoproteins, limited information is available concerning the kinases involved or the physiological relevance of their phosphorylation.Phosphorylation by the cyclin-dependent kinase 8 targets NICD for proteasomal degradation [16], while the atypical protein kinase Cζ regulates Notch1 endocytosis and activity [17], but additional Notch kinases are likely to exist. Analogous to Notch proteins, oncogenic Pim family kinases (Pim1-3) contribute to development of both hematopoietic malignancies and solid tumors [18][19][20][21] by supporting cell survival [22,23], promoting cancer cell migration and invasion [24][25][26], and controlling mitochondrial integrity as well as glucose metabolism [27,28].Via phosphorylation, the serine/threoninespecific Pim kinases positively or negatively regulate activities of several cellular or viral transcription factors, including nuclear antigens of tumorigenic herpesviruses [29,30].Interestingly, these viral factors do not directly bind to DNA, but control gene expression by hijacking the transcriptional machinery of their host cells and by interacting with the Notch coactivator CSL [31], raising the question of whether Pim kinases can also directly or indirectly modulate Notch signaling. In this report, we demonstrate that Pim kinases phosphorylate Notch1 on Serine 2152 within the intracellular domain, and thereby enhance the nuclear localization and activity of Notch1.This crosstalk between Pim and Notch proteins enhances tumorigenic growth of breast and prostate cancer cells via cell type-specific effects, by balancing breast cancer cell metabolism and by promoting prostate cancer cell motility, respectively. ", "section_name": "Research Paper", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "To assess the putative effects of Pim kinases on Notch activity, we used cancer cell lines, which endogenously express both Pim and Notch family members.Western blot analyses showed that Notch1 and Notch3 as well as all three Pim family kinases are expressed in MCF-7 breast cancer cells (Figure 1A), whereas PC-3 prostate cancer cells express all but Notch3.Endogenous Notch activity was then measured by CSLdependent luciferase reporter assays from MCF-7 cells, which had been transfected with previously validated reagents [24] to decrease or increase Pim expression or activity.RNA interference oligonucleotides targeting individual Pim family members reduced reporter activity, while ectopic overexpression of Pim1 enhanced it (Figure 1B-1C).The kinase activity of Pim1 was essential for its enhancing effects, since a kinase-deficient (KD) mutant of Pim1 remained ineffective (Figure 1C).In addition, Notch reporter activity was reduced by two structurally unrelated Pim-selective inhibitors, DHPCC-9 and SGI-1776 (Figure 1D).Reporter activity was inhibited also in PC-3 cells treated with DHPCC-9 (Figure 1F), while the constitutive activity of a CMV-driven control reporter was not decreased by Pim inhibitors in either cell line (Figure 1E,1G).These results confirm that the negative effects of the inhibitors on the CSL-dependent reporter activity are specific and not simply due to cytotoxicity.In sum, these data indicate that Pim kinase expression and activity enhance endogenous Notch activity. To further explore the physiological relevance of the observed crosstalk between Pim and Notch in cancer, we searched for correlations between their mRNA levels in primary breast and prostate cancer samples using the MediSapiens database.These analyses revealed positive Pearson correlations between PIM1 and NOTCH1 in both types of cancer as well as between PIM1 and NOTCH3 in breast cancer (Supplementary Figure 1A-1B).By contrast, no correlations were found between PIM1 and NOTCH3 in prostate cancer or between PIM1 and NOTCH2 in breast cancer (Supplementary Figure 1A-1B). ", "section_name": "Pim kinases upregulate endogenous Notch activity", "section_num": null }, { "section_content": "Since Pim kinases increased and Pim inhibition reduced Notch activity, we next addressed whether Pim kinases directly target Notch ICDs.Glutathione S-transferase (GST)-tagged NICDs were subjected to in vitro kinase assays with GST-Pim1.Interestingly, Pim1 phosphorylated Notch1 and Notch3, but not Notch2 ICD (Figure 2A), which was in line with the observed Pearson correlations (Supplementary Figure 1).As expected, DHPCC-9 treatment reduced Pim1mediated phosphorylation (Figure 2A), while the inactivating mutation in Pim1 KD completely abolished it (Supplementary Figure 2A). To verify that Pim kinases can phosphorylate Notch1 in cells, we used a stable MCF-7/N1ΔE cell line, where a membrane-tethered, ligand-independent form of Notch1 (N1ΔE) is expressed in a doxycycline-inducible fashion and processed by the endogenous γ-secretase to generate N1ICD.MCF-7/N1ΔE cells were treated with doxycycline and DMSO or DHPCC-9, after which N1ICD was immunoprecipitated and its phosphorylation status analysed by Western blotting using an antibody recognizing serine or threonine residues phosphorylated by basophilic kinases.DHPCC-9 treatment reduced phosphorylation of N1ICD and thereby also increased its gel migration (Figure 2B). Using mass spectrometry, we identified the serine residue 2152 as the major Pim1 target site in Notch1 (Supplementary Figure 2B-2C).The amino acid sequence around S2152 (K-A-R-K-P-S-T) shares high complementarity with the Pim1 consensus sequence K/R-K/R-R-K/R-X-S/T-X´, where X´ is defined as an amino acid with neither a basic nor a large hydrophobic residue chain [32].However, in silico analysis suggested another putative site at S2173 with a similar complementarity to Pim target sequence (A-R-R-K-K-S-Q).Therefore, site-directed mutagenesis was used to replace either S2152 or S2173 with an alanine residue to generate phosphodeficient mutants.Results from in vitro kinase assays revealed that S2152, but not S2173 in N1ICD is phosphorylated by all three Pim kinases (Figure 2C).Serine 2152 is localized in the N1ICD within a potential phosphorylated domain (PPD) at the second nuclear localization signal (NLS) (Figure 2D).When a sequence comparison between Notch family members was performed, mouse and human Notch1 showed high complementarity at the amino acid sequence around S2152 (Supplementary Table 1).For further analyses, we generated a phosphomimicking mutant, where the serine residue was replaced with glutamic acid.From here on, the phosphodeficient mutant is denoted as SA (Notch1 S2152A) and the phosphomimicking mutant as SE (Notch1 S2152E). ", "section_name": "Pim kinases phosphorylate Notch1 at serine 2152 in the intracellular domain", "section_num": null }, { "section_content": "To explore the functional consequences of Pimmediated phosphorylation of Notch1, we generated constructs expressing RFP-tagged Pim1 and GFP-tagged Notch1ΔE wild-type or phosphomutant proteins and transiently overexpressed combinations of them in PC-3 cells.When we analysed the localization of GFP-tagged proteins in these cells, both the wild-type Notch1 protein and the SE mutant mainly localized in the nuclei, while significantly fewer GFP-positive nuclei were observed in cells expressing the SA mutant (Figure 3A-3B).Treatment with the Pim inhibitor DHPCC-9 similarly decreased the presence of wild-type Notch1 in the nuclei, while the γ-secretase inhibitor DAPT completely blocked the cleavage-dependent nuclear translocation of Notch1.Furthermore, the effects of DAPT could not be rescued by coexpressed Pim1.Western blotting assays verified the expression levels for fluorescent Notch1 and Pim1 proteins (Figure 3C). Supporting results were obtained with CSLdependent luciferase reporter assays with untagged Notch1 proteins.Ectopic expression of either wild-type N1ΔE or the SE mutant resulted in vastly elevated luciferase levels as compared to untransfected PC-3 or MCF-7 cells (Figure 3D-3E).By contrast, the transcriptional activity of the SA mutant was remarkably reduced.In both types of cells, treatments with either DHPCC-9 or DAPT efficiently reduced Notch1-induced reporter activity (Figure 3F and data not shown).Collectively, these data suggest that Pim-mediated phosphorylation enhances both the nuclear localization and transcriptional activity of Notch1. ", "section_name": "Phosphorylation at Pim target sites increases Notch1 nuclear localization and activity", "section_num": null }, { "section_content": "We next assessed the physical interactions between fluorescently tagged Pim1 and N1ICD by confocal and fluorescence-lifetime imaging microscopy (FLIM).Confocal microscopy revealed that Pim1 colocalized with both wild-type and phosphomutant N1ICD within the nuclei in PC-3 cells (Figure 4A).Furthermore, significantly reduced GFP lifetimes were observed by FLIM when Pim1 and N1ICD were co-expressed, indicating that these proteins physically interact with each other (Figure 4B-4C).By contrast, this interaction was lost, when cells were treated with DAPT to prevent the cleavage and nuclear translocation of N1ICD (Figure 4B). Additional fluorescent assays were carried out to confirm that also endogenously expressed Pim1 and Notch1 proteins are able to colocalize and interact with each other.Staining of MCF-7 cells with Pim1-specific antibodies revealed that endogenous Pim1 is distributed throughout the cells in both the nucleus and the cytoplasm, but not in the nucleoli (Figure 5A).By contrast, staining with Notch antibodies that recognize both membrane-bound and cleaved Notch1 indicated that in non-stimulated MCF-7 cells, endogenous Notch1 mostly resides on the plasma membrane (Figure 5A).Thus, there was only a small fraction of N1ICD that was able to enter the nucleus and activate transcription there, as was also evident from our luciferase results (Figures 1 and3). The endogenous interactions of Pim1 and Notch1 were confirmed from MCF-7 cell samples by in situ proximity ligation assays (PLA; Figure 5B).Strong cytoplasmic signals were observed between Pim1 and Notch1, but not by Notch1 alone or in combination with cytochrome oxidase II (CoxII), which were used as negative controls.The cytoplasmic colocalization and interaction patterns of endogenously expressed Pim1 and Notch1 suggest that Pim1 phosphorylates Notch1 already in the cytoplasm and thereby promotes its nuclear translocation, as supported by the data with ectopically expressed proteins. ", "section_name": "Pim1 colocalizes and interacts with Notch1", "section_num": null }, { "section_content": "To determine whether there is reciprocal regulation of Pim kinases by Notch proteins, we analysed the effects of Notch1 on Pim protein levels in MCF-7 and PC-3 cells.Since Pim kinases are constitutively active whenever expressed [33], changes in their expression levels are expected to directly correlate with their activities.Doxycycline-inducible overexpression of N1ΔE in stably transfected MCF-7 cells resulted in slightly upregulated expression of Pim family members, while more significant increases in them were observed after transient overexpression of wild-type Notch1 in PC-3 cells (Figure 6A-6B).Interestingly, the SA mutant was unable to upregulate Pim expression (Figure 6B), suggesting that phosphorylation-dependent Notch activity is necessary for the observed increase in Pim expression.Accordingly, DAPT treatment also reduced Pim protein levels in untransfected PC-3 cells (Figure 6C).By contrast, the Pim inhibitor DHPCC-9 had no effects on the endogenous expression of N1ICD, when analysed with an antibody recognizing only the cleaved form of Notch1 (Figure 6D). ", "section_name": "Pim protein levels are upregulated by Notch1", "section_num": null }, { "section_content": "Both Pim kinases and Notch1 have been shown to promote cancer cell migration and invasion [24,34].To determine whether there is a hierarchical or synergistic relationship between these proteins, we performed wound healing assays in PC-3 cells.Inhibition of endogenous Notch activity by DAPT decreased cell migration to the same extent as Pim inhibition by DHPCC-9, even when Pim1 was overexpressed (Figure 7A).Conversely, activation of endogenous Notch signaling by immobilized Jagged1 ligand enhanced migration, which was antagonized not only by DAPT, but also by the Pim inhibitor DHPCC-9 (Figure 7B).Transient overexpression of wild-type N1ΔE or the SE mutant similarly increased cell migration, whereas overexpression of the SA mutant was unable to do so (Figure 7C).Furthermore, the SE mutant was even able to partly rescue the negative effects of the Pim inhibitor DHPCC-9 on cell motility.Taken together, these data indicate that PC-3 cells need activities of both Pim kinases and Notch1 for efficient cell migration. To confirm that the increase in PC-3 cell migration was not simply due to enhanced cell proliferation, we used IncuCyte analyses to measure cell confluency at several time-points after transfection.Indeed, during the 72 h follow-up period, it became evident that overexpressed Notch1 and Pim1 proteins reduce rather than increase the ability of cells to reach confluency (Supplementary Figure 3A-3B). ", "section_name": "Pim-mediated cell migration is dependent on Notch1 phosphorylation and activity", "section_num": null }, { "section_content": "Both Pim kinases and Notch1 have been implicated in the control of glucose metabolism [9,28].In breast cancer cells, Notch activation induces a glycolytic switch, while Notch inhibition leads to defects in mitochondrial function and a forced glycolytic phenotype [9].To evaluate the role of Pim kinases and their potential interplay with Notch1 in breast cancer cell metabolism, MCF-7 cells were treated with the Pim-selective inhibitors DHPCC-9 or SGI-1776.Inhibition of Pim activity increased glucose uptake in association with a higher mitochondrial membrane potential (Figure 8A-8B), which was indicative of defects in mitochondrial function.In a similar fashion, the N1ΔE SA mutant increased glucose uptake as compared to the wild-type Notch1 protein or the SE mutant (Figure 8C).When lactate production was measured as the endpoint of enforced glycolytic metabolism, both Pim inhibition by DHPCC-9 and overexpression of the N1ΔE SA mutant increased lactate levels, while the SE mutant partially rescued the effects of DHPCC-9 (Figure 8D).Conversely, overexpression of Pim1 had opposite effects (Figure 8E), supporting efficient utilization of internalized glucose for OXPHOS and protein synthesis, as previously reported [28].Taken together, these data suggest that Pim1 counteracts the effects of Notch1 in regulation of breast cancer cell metabolism. IncuCyte and MTT assays were performed to exclude the possibility that our results were affected by the influence of Pim or Notch upregulation or inhibition on MCF-7 cell growth rate or survival, respectively.In IncuCyte analyses, neither Notch1 nor Pim1 increased the ability of cells to reach confluency (Supplementary Figure 3C-3D).In addition, no significant differences were observed in the viabilities of cells transfected with wild-type or mutant N1ICDs or treated with DAPT or DHPCC-9 (Supplementary Figure 3E-3F).These MTT assay results are in line with our previously published data, according to which DHPCC-9 does not affect viability of PC-3 cells [24]. ", "section_name": "Crosstalk between Notch1 and Pim1 regulates breast cancer cell metabolism", "section_num": null }, { "section_content": "Finally, we probed the possible synergistic effects of Pim and Notch activity on tumor growth.For this purpose, we used the well-established chick embryo chorioallantoic membrane (CAM) xenograft model [35], which we have previously used to assess the effects of Notch inhibition on breast and prostate cancer growth [36].MCF-7 breast cancer cells transiently overexpressing N1ΔE wildtype or phosphomutants were xenografted onto CAM, and tumor growth was followed for 5 days.Part of the samples was treated with estradiol, which has been shown to upregulate Pim1 expression in the hormone-dependent MCF-7 cells [37].In the presence of estradiol, wild-type Notch1 and the SE mutant both enhanced tumor growth, while the SA mutant strongly suppressed it (Figure 9A).Furthermore, the Pim inhibitor DHPCC-9 efficiently reduced Notch1-induced tumor growth to the same level as the SA mutation.In the absence of estradiol, the tumorpromoting effect of Notch1 was almost lost (Figure 9B).Intriguingly, already nanomolar concentrations of estradiol upregulated Pim1 and Pim3, but not Pim2 protein levels in cultured MCF-7 cells (Figure 9C), further supporting the conclusion that their expression is essential for full Notch1 activity. We also examined the effects of Pim and Notch inhibitors on the growth of untransfected PC-3 cells on CAM.Pim inhibition by DHPCC-9 or blockade of Notch signaling by DAPT efficiently reduced tumor volume, while the most pronounced tumor-suppressive effects were obtained by a combinatorial treatment (Figure 9D).Furthermore, DAPT treatment was also able to decrease growth of tumors formed by PC-3 cells stably overexpressing Pim1 (Supplementary Figure 4). To further verify the cross-talk between Pim and Notch proteins in prostate cancer, we analysed the presence of nuclear Notch1 in orthotopic prostate xenografts.We have previously shown that Pim overexpression induces metastatic growth of PC-3-derived orthotopic xenografts in mice [25].Immunostaining of the xenografted samples showed enhanced numbers of N1ICD-positive nuclei in tumors with stable Pim1 or Pim3 overexpression, while mock-transfected xenografts or Pim3-overexpressing xenografts from mice treated with the Pim inhibitor DHPCC-9 displayed fewer N1ICD-positive nuclei (Figure 9E-9F).This is in line with the observed changes in N1ICD localization in cultured PC-3 cells after Pim inhibition or overexpression of the phosphodeficient Notch1 mutant (Figure 3A-3B).These data together with the CAM results suggest that Pim/Notch crosstalk drives tumor progression (Figure 10), and that combinatorial therapies might be beneficial for cancer patients with deregulated expression of both Pim and Notch proteins. ", "section_name": "Notch1 and Pim kinases synergize to promote tumor growth in vivo", "section_num": null }, { "section_content": "In this report, we have unraveled a novel link between Pim kinases and Notch1, which is relevant for the progression of both breast and prostate cancer.We show that all three Pim family kinases phosphorylate the intracellular domain of Notch1 (N1ICD), and thereby stimulate the nuclear localization and transcriptional activity of N1ICD.Pim kinases also phosphorylate Notch3, but not Notch2.Notch4 lacks Pim consensus sites and is thus not expected to be a Pim substrate.Here we focused on the phosphorylation of Notch1, as expression of Notch 3 was restricted to MCF-7 breast cancer cells and not present in PC-3 prostate cancer cells, and the exact role of Notch3 in tumor progression is still under debate [38,39].The Pim1 phosphorylation target site at S2152 resides in a linker between two clusters of basic amino acids, the entire sequence of which has been observed to contain a nuclear localization signal (NLS) for N1ICD [40].N1ICD has been recognized as a target for the nuclear transport receptor importin-α, [41], and the linker sequence between the basic amino acid clusters has been proposed to facilitate direct association with importin-α [42,43].Our findings support this hypothesis and suggest that nuclear transport is associated with phosphorylation of S2152. Pim-induced increase in the nuclear localization of N1ICD is observed in both cultured cells and in orthotopic prostate cancer xenografts, while Pim inhibitors or mutagenesis of the phosphorylation target site have opposite effects.The importance of our data is further enforced by the conservation of the Pim target site in Notch1 across several animal species [44].Phosphorylation is not essential for the ability of Pim1 and Notch1 to interact with each other, as both wildtype Notch1 and phosphodeficient or phosphomimicking mutants all colocalize and physically interact with Pim1.This correlates with our previous observations demonstrating that both wild-type and kinase-deficient Pim1 can interact with Pim substrates [23,45].Our data indicate that endogenous Pim1 and Notch1 interact already in the cytoplasm, which supports the notion that Pim1 enhances nuclear localization and activity of Notch1. Phosphorylation of Notch1 by Pim kinases promotes motility of prostate cancer cells, as demonstrated both by the phosphomutants and by the ability of either Pim or Notch inhibitors to block the pro-migratory effects of Notch or Pim proteins, respectively.In breast cancer cells, Pim-mediated phosphorylation of Notch1 balances cell metabolism, while its inhibition enforces glycolytic metabolism via defects in mitochondrial function, as previously shown for Notch inhibition [9].This conclusion is supported by the reported abilities of Pim kinases to preserve mitochondrial integrity [27] and to regulate glycolysis and mitochondrial biogenesis by influencing expression of PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) and c-Myc [28], which also is a transcriptional target for Notch1 [46].Similar metabolic effects were not observed in prostate cancer cells, which are energetically less dependent on glycolysis [47]. The impact of Pim-mediated phosphorylation of Notch on tumor progression is demonstrated by our in vivo data from the CAM assays, where Pim and Notch synergistically enhance tumorigenic growth of both breast and prostate cancer cells.Treatment with the Pim inhibitor DHPCC-9 efficiently blocks the tumor-promoting effects of Notch1, and vice versa, the γ-secretase inhibitor DAPT abrogates the tumor-promoting effects of Pim1.Furthermore, we show that simultaneous inhibition of both Pim and Notch activities more efficiently inhibits tumor growth than targeting either one alone. Since the Pim inhibitor DHPCC-9 and the S2152A mutation in Notch1 reduce Notch activity and tumor growth to a similar extent, this suggests that Pim kinases are the major kinases targeting Ser2152 of Notch1 in both MCF-7 and PC-3 cells.However, it remains possible that also other kinases such as Akt target it, since Akt is known to share some but not all substrates with Pim kinases [48].Interestingly, we observed that up-or downregulation of Notch1 activity correlates with increased or decreased Pim expression levels, respectively.Thus, there may be a positive feedback loop, through which phosphorylation of Notch1 by Pim1 results in increased Pim levels to further enhance Notch1 activity, but not Notch1 expression. Since Pim kinases and Notch1 play important functions in tumorigenesis, it is not surprising that there are major efforts underway to target their activities for cancer therapy [1,19,21].We show that inhibition of Pim-mediated phosphorylation of Notch1 efficiently reduces tumor growth, and that simultaneous inhibition of Notch and Pim is even more effective.Our data suggest that the synergy between Pim and Notch leads to a more malignant behavior.Hence, combinatorial targeting of Pim and Notch proteins or their downstream targets may provide novel and effective approaches for cancer therapy. ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "Human PC-3 prostate cancer and MCF-7 breast cancer cell lines and their derivatives were cultured as previously described [9,24,25].Stable control or human Pim1-overexpressing PC-3 cell lines were cultured in the presence of 200 μg/ml G418.For endogenous Notch activation, PC-3 cells were cultured on plates treated with 50 μg/ml of protein G/PBS overnight, 10 mg/ml BSA/PBS for 1 h and finally recombinant 2 μg/ml of Jagged1-FC or control FC as previously described [17].To induce Notch1 expression in the stable MCF-7/N1ΔE cell line, 1 μg/ml of doxycycline (Sigma-Aldrich, St. Louis, MO, USA) was used.Pim kinase activity was inhibited by DHPCC-9 (1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde; [24,49]) or SGI-1776 (N-[(1-methylpiperidin-4-yl) methyl]-3-[3-(trifluoromethoxy)phenyl]imidazo[1,2-b] pyridazin-6-amine; S2198, SelleckChem, Houston, TX, USA), while the γ-secretase inhibitor DAPT (N-[N-(3,5difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; Calbiochem, San Diego, CA, USA) was used to prevent Notch cleavage and nuclear entry.The inhibitors were diluted in DMSO, which was also used as a control (maximum concentration in cell culture 0.1%).Various concentrations of estradiol (E2) (E8875, Sigma-Aldrich) were used for treatment of cultured or xenografted MCF-7 cells.MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich) was used to measure cell viability. ", "section_name": "Cell culture and chemical compounds", "section_num": null }, { "section_content": "Human PIM cDNAs were PCR-cloned from a human kinome cDNA collection [50] to the eukaryotic expression vector pcDNA™3.1/V5-His-C(Invitrogen, Carlsbad, CA, USA).Following primers were used for cloning: For 5'-C CCA AGC TTG ACC ATG CTC TTG TCC AAA ATC AAC-3' and Rev 5'-CAG AAT TCC TTT GCT GGG CCC C-3' (PIM1); For 5'-C CCA AGC TTG ACC ATG TTG ACC AAG CCT CTA CAG-3' and Rev 5'-CAG AAT TCC GGG TAG CAA GGA CCA GG-3' (PIM2); For 5'-C CCA AGC TTG ACC ATG CTG CTC TCC AAG TTC G-3' and Rev 5'-CAG AAT TCC CAA GCT CTC GCT GCT GG-3' (PIM3).Human PIM1, PIM2 and murine pim3 (a kind gift from A. MacDonald, University of Dundee, Dundee, UK) were further cloned to pGEX-6P-1 vector (GE Healthcare Life Sciences, Little Chalfont, UK) for GST fusion protein production.Following primers were used for cloning: For 5'-GCC GAA TTC ATG CTC TTG TCC-3' and Rev 5'-GGG GTC GAC CTA TTT GCT GGG CC-3' (PIM1); For 5'-GCC GGA TCC ATG TTG ACC AAG CC-3' and Rev 5'-GGC GTC GAC TTA GGG TAG CAA GG-3' (PIM2); For 5'-GCC GAA TTC ATG CTG CTG TCC-3' and Rev 5'-GCC CTC GAG TCA CAA GCT CTC ACT GC-3' (pim3).Human PIM1 cDNA without a Stop codon and with a Kozak sequence (Genewiz Inc., South Plainfield, NJ, USA) was cloned into the pTagRFP-N vector (FP142; Evrogen, Moscow, Russia). To prepare kinase-deficient (KD) human Pim mutants, the ATP-binding lysines were converted into methionines using the QuikChange site-directed mutagenesis kit (Stratagene, Agilent Technologies, Santa Clara, CA, USA).Following primers were used for mutagenesis: For 5'-GTG GCC ATC ATG CAC GTG GAG AAG GAC CGG ATT T-3' and Rev 5'-CTC CAC GTG CAT GAT GGC CAC CGG CAA GTT G-3' (Pim1 K67>M); For 5'-GTG GCC ATC ATG GTG ATT CCC CGG AAT CGT GTG-3' and Rev 5'-GGG AAT CAC CAT GAT GGC CAC CTG GAG TCG ATC TG-3' (Pim2 K61>M); For 5'-GTG GCT GTG ATG CAC GTG GTG AAG GAG CGG GT-3' and Rev 5'-CAC CAC GTG CA TCA CAG CCA CCG GGA GCC C-3' (Pim3 K69>M). Following constructs were used for overexpression of murine Notch intracellular domains: N1ICD-pGEX-4T-3, N1ΔE-pCS2+ and GFP-N1ΔE [46].N2ICD and N3ICD were cloned to pGEX-6P-3 by cutting the ICDs from the corresponding p3xFlag-CMV-7.0 constructs [51].Site-directed mutagenesis of NICDs was performed by Stratagene Ultra Pfu DNA polymerase.Following primers were used for mutagenesis: For 5'-GCC ACA CAG GGA AAG AAG GCG CGC AAG CCA GC T ACC AAA GGG C-3' and Rev 5'-G CCC TTT GGT AGC TGG CTT GCG CGC CTT CTT TCC CTG TGT GGC-3' (Notch1 S2152>A, \"SA\"); For 5'-G GAC CTC AAG GCG CGC AGG AAG AAG GCA CAG GAT GGC AAG GGC-3' and Rev 5'-GCC CTT GCC ATC CTG TGC CTT CTT CCT GCG CGC CTT GAG GTC C-3' (Notch1 S2173>A); For 5'-CC ACA CAG GGA AAG AAG GCG CGC AAG CCC GAG ACC AAA GGG-3' and Rev 5'-CCC TTT GGT CTC GGG CTT GCG CGC CTT CTT TCC CTG TGT GG-3' (Notch1 S2152>E, \"SE\"). ", "section_name": "DNA constructs and mutagenesis", "section_num": null }, { "section_content": "Fugene ® 6/HD (Promega, Fitchburg, Wisconsin, USA) 3:1 to DNA was used for PC-3 cell transfections, while MCF-7 cells were transfected either by jetPEI ® (Polyplus Transfection, New York, NY, USA) or by electroporation.For RNA interference, 200 nM nontargeting control siRNA or Pim-targeting siRNA oligonucleotides were transfected by Lipofectamine™ (Invitrogen/Life Technologies, Carlsbad, CA, USA).Following oligonucleotides were used: non-targeting MISSION ® siRNA Universal Negative Control #1, SIC001 (Sigma-Aldrich), D-001810-01-20 ON-TARGETplus Non-targeting siRNA #1, (Dharmacon, Lafayette, CO, USA), 5'-GAU GGG ACC CGA GUG UAU A-3' J-003923-09-0020 ON-TARGETplus PIM1 siRNA (Dharmacon), 5'-GUG GAG UUG UCC AUC GUG ACA UU-3' 5'-UGU CAC GAU GGA CAA CUC CAC UU-3' PIM2 siRNA (Sigma-Aldrich), 5'-GGC GUG CUU CUC UAC GAU AUG UU-3' 5'-CAU AUC GUA GAG AAG CAC GCC UU-3' PIM3 siRNA (Sigma-Aldrich).Notch activity was measured by a 12xCSL luciferase reporter construct, while β-galactosidase was used for normalization as previously described [52].Constitutively active CMVluciferase construct (a kind gift from J. Ivaska, Turku Centre for Biotechnology, Turku, Finland) was used as an additional control. ", "section_name": "Transfections and transactivation assays", "section_num": null }, { "section_content": "GST-tagged fusion proteins were separated from glutathione sepharose beads by 30 mM glutathione in 75 mM Tris-HCl (pH 8.0), thereafter GST-tagged Pim and Notch family members were subjected to similar in vitro kinase reactions as previously described [53].Following kinase assays, mouse N1ICD was subjected to in-gel trypsin digestion and TiO 2 affinity chromatography as previously described [54].Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed using a Q Exactive mass spectrometer (Thermo Fisher Scientific).Database search was performed using Mascot 2.4 (Matrix Science) via Proteome Discoverer 1.3 (Thermo Fisher Scientific) against the Swiss-Prot (E. coli and recombinant proteins) database.Label-free quantification was performed using Progenesis LC-MS 4.0 (Nonlinear Dynamics).Protein sequence comparison was performed by BLAST (Basic Local Alignment Search Tool by The National Center for Biotechnology Information), while the sequences were obtained from Swiss-Prot Universal Protein Resource Knowledgebase. ", "section_name": "In vitro kinase assays, mass spectrometry and in silico analysis", "section_num": null }, { "section_content": "PC-3 cells were plated on cover glasses and transiently transfected with RFP-or GFP-tagged expression vectors.After 48 hours, samples were fixed with 4% paraformaldehyde, washed with PBS and mounted with Mowiol.Samples were imaged by Leica TCS SP1 and TCS SP5 confocal microscopes with HCX PL APO CS 63x1.3Oil objective with LCS 2.61 or LAS AF Application (Leica Microsystems CMS GmbH, Mannheim, Germany).Excitation wavelengths were 488 nm (GFP) and 561 nm (RFP), while emission wavelengths were 500-535 nm (GFP) and 599-651 nm (RFP).Sequential scanning was performed to reduce the background signal in colocalization imaging.Physical interactions between tagged proteins were measured by fluorescence-lifetime imaging microscopy (FLIM) as previously described [55].By this method, fluorescence resonance energy transfer between two closely located fluorophores can be detected by measuring the change in the donor fluorophore (e.g.GFP) lifetime in the presence or absence of the acceptor (e.g.RFP) [56]. To analyse interactions between endogenously expressed Pim1 and Notch1 proteins, in situ proximity ligation assays (PLA) were carried out as previously described [57].For this purpose, MCF-7 cell samples were fixed for 10 min with methanol and 1 min with acetone, and stained with Pim1 (ab117525, Abcam, Cambridge, UK), Notch1 (C20, Santa Cruz Biotechnology, Dallas, TX, USA) or CoxII (12C4, Santa Cruz) primary antibodies.Thereafter, the assays were continued using Duolink DUO92102 reagents (Sigma-Aldrich) according to manufacturers instructions.Finally, samples were mounted with DAPI-containing medium (Duolink DUO82040, Sigma-Aldrich), and imaged using the Zeiss LSM760 (ZEISS, Oberkochen, Germany) confocal microscope. ", "section_name": "Fluorescence microscopy", "section_num": null }, { "section_content": "Western blotting samples were prepared and run as previously described [9].Notch1 immunoprecipitation was performed with anti-cleaved Notch1 SAB4502019 antibody (Sigma-Aldrich) as previously described [17].Primary antibodies from Cell Signaling Technology (Danvers, MA, USA) were diluted 1:1000 (anti-Pim1 #2907; anti-Pim2 #4730; anti-Pim3 #4165; anti-β-actin #4970S, anti-Notch1 Val1744, anti-phospho Ser/Thr (RXXS/T) #9614), while their secondary antibodies were diluted 1:5000 (anti-mouse #7076, anti-rabbit #7074).Other primary antibodies were diluted 1:500 (anti-Pim1 12H8, Santa Cruz Biotechnology) or 1:1000 (anti-Notch1 activated ab8925, Abcam; anti-HSC70/ HSP73 1B5, Enzo Life Sciences, Farmingdale, NY, USA).Amersham™ ECL™ Plus/Prime reagents (GE Healthcare Life Sciences, Little Chalfont, UK) were used for chemiluminescence reactions.Signal intensities from SDS-PAGE and Western blotting samples were analysed by the ChemiDoc™ MP Imaging System with Image Lab software Version 4.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).Relative signal intensities were calculated based on protein loadings or control stainings. ", "section_name": "Protein analyses", "section_num": null }, { "section_content": "A confluent PC-3 cell layer was scratched by a 10 μl pipette tip 24 h after transfection or Notch activation.Wounded cells were either left untreated or treated with DMSO-diluted compounds.Samples were imaged by Olympus CK40 microscope (Olympus Corporation Tokyo, Japan) with 20x enlargement and analySIS getIT 5.0 software (Olympus Soft Imaging Solutions GmbH, Münster, Germany), and analysed as previously described [24]. ", "section_name": "Wound healing assays", "section_num": null }, { "section_content": "IncuCyte™ (Essen BioScience, Ann Arbor, MI, USA) was used for automatic measurement of cell confluency on 96-well plates.Cell viability was measured by MTT assays as previously described [24]. ", "section_name": "Proliferation and viability assays", "section_num": null }, { "section_content": "MCF-7 cells were incubated with 100 μM fluorescent 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), after which cells were detached, washed and analysed by flow cytometer BD FACSCalibur with BD CellQuest PRO v.5.1.1 software (BD Biosciences, San Jose, CA, USA) as previously described [9]. ", "section_name": "Glucose uptake", "section_num": null }, { "section_content": "Flow cytometry was applied for analysis of mitochondrial permeability using the tetramethylrhodamine methyl ester (TMRM) dye as previously described [9]. ", "section_name": "Mitochondrial membrane potential", "section_num": null }, { "section_content": "Conditioned medium was collected and deproteinated with 300 mM perchloric acid for 10 min on ice.After centrifugation, the supernatant was neutralized with 300 mM potassium hydroxide.After another centrifugation, lactate was converted to pyruvate by lactate dehydrogenase in the presence of nicotinamide adenine dinucleotide (NAD) (both from Sigma-Aldrich).Samples were incubated for 1 h at 37°C in a buffer containing 400 mM hydrazine sulphate and 500 mM glycine, after which formation of NADH was measured spectrophotometrically at 340 nm by Envision Multilabel Reader (PerkinElmer, Turku, Finland). ", "section_name": "Lactate production", "section_num": null }, { "section_content": "Using the CAM model [35], 0.5-2 x 10 6 cancer cells were transplanted onto the CAM of fertilized chicken eggs.The cells were allowed to form tumors and become infiltrated with the vasculature of the CAM for 5 days, during which tumors were treated daily with estradiol, DHPCC-9 or DAPT as indicated in the figures.The estradiol concentrations were chosen to be within the same range as previously used in mouse experiments [58].On day 5, the tumors were fixed in ovo with 3% paraformaldehyde for 3h, removed, dehydrated and weighted. ", "section_name": "Chorioallantoic membrane (CAM) model", "section_num": null }, { "section_content": "Samples for immunohistochemical analysis were gained from a previous study [25].Animal experiment procedures were approved by the Provincial State Office of Western Finland with the licence ID ESAVI/3937/04.10.03/2011.Shortly, stable Pim1, Pim3, or empty plasmid (mock) overexpressing PC-3-derived prostate xenografts were allowed to grow for approximately three weeks, while part of the mice were daily treated with 50 mg/kg of the Pim inhibitor DHPCC-9.Antigen retrieval and peroxidase blocking were performed to paraffin-embedded tumor samples as previously described [9].TBS was used instead of PBS.Samples were blocked in Dako Antibody Diluent (S0809, Agilent Technologies, Dako Denmark A/S, Glostrup, Denmark) for 10 min at RT, stained with primary antibody anti-Notch1 (Val1744, Cell Signaling Technology) 1:500 for 1 h at RT and secondary antibody Poly-HRP-Anti-rabbit IgG (DPVR55HRP, Agilent Technologies) for 30 min at RT. DAB treatment and hematoxylin counterstaining have been previously described [9].Whole tumor scanning was performed as previously reported [25].Double blind analysis were performed manually, and necrotic areas were left out from analysis. ", "section_name": "Immunohistochemistry", "section_num": null }, { "section_content": "Bar graphs were produced by Microsoft Excel 2013 or GraphPad Prism 4.00 and results were analysed by Student's t-test and ANOVA.Pearson correlations for mRNA levels were obtained from the MediSapiens database (medisapiens.com).In each analysis, P<0.05 was used as a limit for significant difference.Error bars represent standard deviations.Figures were prepared by Corel Draw X5 or Adobe Illustrator CS5 15.0.0. ", "section_name": "Statistical analysis and figure preparation", "section_num": null } ]	[ { "section_content": "We thank R. Arnaudova, P. Haapaniemi, A. Heinonen J. Korhonen, S. Kollanus, J. Kujala, M. Lagström, J. Sandholm, H. Siimesvaara, E. Siljamäki, R. Vahakoski and E. Özliseli for technical assistance, and A. McDonald and J. Ivaska for reagents.We are grateful for obtaining DHPCC-9 from F. Anizon and P. Moreau (Clermont Université, Université Blaise Pascal and CNRS, France).www.impactjournals.com/oncotargetFrenckell Foundation, K. Albin Johansson Foundation, Liv och Hälsa Foundation, Knut and Alice Wallenberg Foundation, the Swedish Cancer Society, the Swedish Research Council, EU (ITN NotchIT) and Karolinska Institutet (BRECT Theme Center) (SKJL, UL). ", "section_name": "ACKNOWLEDGMENTS", "section_num": null }, { "section_content": "The authors declare no conflicts of interest. ", "section_name": "CONFLICTS OF INTEREST", "section_num": null }, { "section_content": "", "section_name": "GRANT SUPPORT", "section_num": null } ]
10.1186/s40164-024-00541-3	1q jumping translocation as a biomarker in myeloid malignancy: frequently mutated genes associated with bad prognosis and low survival	<jats:title>Abstract</jats:title><jats:p>1q jumping translocation (JT) is rare and its molecular profiles in myeloid malignancies are not well-known. This study evaluated gene mutations in 1q-JT cohorts (0.38%) from hematological malignant specimens that underwent genetic analysis at the Johns Hopkins Hospital (<jats:italic>n</jats:italic> = 11,908) and the MD Anderson Cancer Center. 1q-JT had frequent mutations in eleven genes, most of which are associated with worse prognosis. <jats:italic>BCOR</jats:italic> mutations significantly co-occurred with others. Patients tended to have mutations in DNA-repair, spliceosome, and epigenetic modification pathways, though genes utilized within each of these pathways were not randomly distributed. Multi-, albeit overlapping, pathway interruptions tended to manifest in mutations of two gene sets. One gene set consisted of <jats:italic>SF3B1</jats:italic> (spliceosome) and <jats:italic>TET2</jats:italic> (epigenetic modification), while the other consisted of <jats:italic>STAG2</jats:italic> (DNA repair), <jats:italic>SRSF2</jats:italic>, <jats:italic>U2AF</jats:italic> (spliceosome), <jats:italic>ASXL1</jats:italic>, <jats:italic>KMT2D</jats:italic> (epigenetic modification), <jats:italic>BCOR</jats:italic>, and <jats:italic>GATA2</jats:italic> (transcription factors). An “intermediate” JT-like rearrangement may represent an early sign of occurring 1q-JT. Treatments (hypomethylating agents) and unique structures of the short arms of acrocentric chromosomes may contribute to 1q-JT formation in myeloid malignancies. The median overall survival after identification of a JT was 10 months (95% confidence interval, 5–15 months). Our cohort represents the largest number of myeloid malignancies from multi-centers with before and after the 1q-JT event analyzed to date. Overall, this study identified specific molecular profiles that are associated with 1q-JT in myeloid malignancies. 1q-JT could serve as a poor prognosis biomarker in myeloid malignancies, which could be important in making well-informed clinical decisions and treatment strategies.</jats:p>	[ { "section_content": "1q jumping translocation as a biomarker in myeloid malignancy: frequently mutated genes associated with bad prognosis and low survival Eitan Halper-Stromberg 1 , Victoria Stinnett 2 , Laura Morsberger 2 , Aparna Pallavajjala 2 , Mark J. Levis 2 , Amy E. DeZern 2 , Michelle Lei 2 , Brian Phan 2 , Rena R. Xian 2 , Christopher D. Gocke 2 , Guilin Tang 3 and Ying S. Zou 2* ", "section_name": "", "section_num": "" }, { "section_content": "Jumping translocation (JT) is a rare chromosomal rearrangement comprising one donor and multiple recipient chromosomes [1].JTs involve bands 1q12-q21 as the donor segment, referred to as 1q-JT, which have been reported in ~ 50 myeloid malignancies and only a few patients had mutation data [2][3][4][5].Given the rarity of 1q-JTs in myeloid malignancies and lack of large case series, the molecular profiles of 1q-JT cases in myeloid malignancies are not well-known. We reviewed 11,908 hematological malignant specimens that underwent karyotyping performed from 2016 to 2023.46 (~ 0.38%) specimens from 21 patients had myeloid malignancies and 1q-JT (Table 1, S1, Fig. 1A).Across 56 specimens (19 pre and 37 post JT) from 20 patients with concurrent deep Next-Generation-Sequencing (NGS, supplementary methods [6]), we observed 45 mutated genes (Table S2).Eleven of 45 (24%) were mutated in ≥ four samples (Fig. 1B).Mutations in eight genes (SRSF2, ASXL1, KMT2D, GATA2, U2AF1, SF3B1, BCOR, and STAG2) were enriched in our samples (P value < 0.05 based on a chi square test) compared with mutation frequencies from the Beat AML study [7].These include multiple genes that are associated with worse prognosis (SRSF2, U2AF1, KMT2D, ASXL1, RUNX1, BCOR, TET2). Among the most frequently mutated genes, the most frequently significantly co-mutated gene was BCOR (Fig. 1C, Pearson correlation tests).BCOR was significantly co-mutated with KMT2D and STAG2, q-value = 0.01 (Fig. 1C).Two other pairs of genes were significantly co-mutated, one pair is U2AF1 and GATA2 and the other pair is RUNX1 and SRSF2 (Fig. 1C).SRSF2 and RUNX1 have been shown previously to co-mutate in AML [8]. Across the thirteen patients with specimens tested both before and after the JT event, the median variant allele frequencies (VAFs) of mutations in these eleven genes did not change significantly post-JT (P value 0.21 -1.0 based on Wilcoxon rank sum test). For validation of our mutational findings within the Johns Hopkins (JH) cohort, we collected an additional 25 patients with myeloid malignancies from MD Anderson (MDA) including 18 1q-JT patients and 7 non-1q JT patients (Table 1, S1).Of the 45 mutated genes from the JH-cohort, 17 were observed to be mutated in ≥ one patient in the MDA-cohort.Of the eleven genes most mutated in the JH-cohort, eight were included in the MDA-panel and five were mutated in ≥ two patients in the 1q-JT MDA-cohort (Figure S1).In descending order of frequency, these genes included RUNX1, SRSF2, TET2, SF3B1, and ASXL1. Even with a large number of simple/non-complex karyotypes (93.5%) in this study (Table 1/S1), the median overall survival after JT occurrence was 10 months (Fig. 1D,S2).Longer survivals were observed in patients with allogeneic hematopoietic cell transplantation (alloHCT). Pre 1q-JT patients had a long interval [median = 914day, range = 105-7539 days] to the first specimens with 1q-JT occurrence from diagnosis (Table S1).The majority of patients had treatments involving hypomethylating agents (HMAs) before JT (Figure S2).For example, case #2 had maintenance azacitidine for > 9-year after alloHCT before he developed 1q-JT.He and other two patients (case #17, 36) also developed an \"intermediate\" JT-like chromosomal rearrangement, with 1q donor to only one recipient chromosome.Some mutations, such as BCOR and TET2, have been reported to be associated with being sensitive and having a better response to HMAs [8,9].HMAs might be associated with hypomethylating the large pericentromeric heterochromatin region of chromosome 1 [10,11]. SNP microarray, optical genome mapping, and the NGS data revealed 1q-gain with various proximal breakpoints and the same terminal breakpoint and either one or both homologous chromosomes 1 as JT donor chromosomes (Table S3, Figures S3-5).Except two terminal deletions of two recipient chromosomes (Telomeric 1pter and 18pter regions involving in 1q-JT in case #16), no additional deletions among recipient chromosomes were observed by SNP microarray or optical genome mapping.Combination 1q-gain and the pre-JT gene mutations may cooperate to promote cancer progression and develop more aggressive disease. This study is the first multi-center cohort having mutation profiles of before and after the 1q-JT events in myeloid malignancies.This study revealed frequent mutations, most of which are associated with worse prognosis.While pre 1q-JT had a long > 2.5-year median interval to the specimens with 1q-JT occurrence, the median overall survival after 1q-JT was 10 months, supporting combination 1q-gain and the pre-JT gene mutations may cooperate to develop more aggressive disease.To our knowledge, our cohort represents the largest number of 1q-JT in myeloid malignancies analyzed to date.Overall, 1q-JT could serve as a poor prognosis biomarker in myeloid malignancies.1B) and the other tending towards mutations in BCOR, KMT2D, STAG2, SRSF2, RUNX1, U2AF1, GATA2, and ASXL1 (Fig. 1B), though mutations were not mutually exclusive across groups.Specimens lacked mutation in NPM1, a frequently mutated gene in AML, associated with better prognosis.FLT3-ITD, a mutation associated with worse prognosis, was detected in one of the 20 patients.(C) Among the most frequently mutated genes, the most frequently significantly co-mutated gene (q-value < 0.05 based on Benjamini-Hochberg false discovery rate control of P-values from Pearson correlation tests) was BCOR.BCOR was significantly co-mutated with KMT2D, q-value = 0.01, and STAG2, q-value = 0.01 (numbers in circles indicate Pearson correlation coefficients, values obscured with an 'X' have P-values >=0.05).Two other pairs of genes were significantly co-mutated, U2AF1 and GATA2 q-value = 0.01, and RUNX1 and SRSF2, q-value = 0.04.(D) Diagram of potential mechanisms associated with development of 1q-JT in myeloid malignancies.Pre 1q JT patients with myeloid malignancies had these frequent mutations and may treat with hypomethylating agents (HMAs) such as azacitidine and decitabine before JT.HMAs may lead to epigenetic alteration such as hypomethylation of chromosome 1q12 pericentric heterochromatin, which may contribute to development of a double-strand break.Short arms of acrocentric chromosomes are frequently involved in 1q-JT formation because their distinctive genomic structure with centromere sequences and no coding genes makes them well-known for chromosomal rearrangements/ recombination.The presence of these pre JT mutations and gain of 1q due to JT led to a poor survival in these 1q-JT patients with myeloid malignancies.The median overall survival after JT occurrence was 10 months (95% confidence interval, 5-15 months) in this study ", "section_name": "To the editor", "section_num": null }, { "section_content": "", "section_name": "Table 1 Jumping translocation (JT) cohorts in this study", "section_num": null } ]	[ { "section_content": "The authors are grateful to all personnel of the Johns Hopkins Cytogenomic Laboratory, Johns Hopkins Genomics, and the University of Texas MD Anderson Cancer Center involved in genetic testing.This study was supported by the Johns Hopkins School of Medicine Department of Pathology. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "This work was supported by the Johns Hopkins School of Medicine Department of Pathology. ", "section_name": "Funding", "section_num": null }, { "section_content": "No datasets were generated or analysed during the current study. ", "section_name": "Data availability", "section_num": null }, { "section_content": "The online version contains supplementary material available at https://doi.org/10.1186/s40164-024-00541-3. Additional file 1. Supplementary data includes supplementary material and methods, supplementary tables S1-2 and supplementary figure S1-S4 Author contributions EH, LM, AP, GT, and YZ performed study concept and design; EH and YZ performed writing; all authors provided acquisition, analysis and interpretation of data, EH performed statistical analysis; BP, ML, LM, and AP provided technical and materials support.EH, LM, and YZ contributed to review and revision.EH, VS, LM, AP, MT, AD, ML, BP, RX, CG, GT, and YZ read and approved the final paper. This study was approved by the Institutional Review Board (JHIRB#00278530) and performed in accordance with the Declaration of Helsinki. A retrospective study and the informed consent was waived by the Institutional Review Board. The authors declare no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Supplementary Information", "section_num": null }, { "section_content": "The online version contains supplementary material available at https://doi.org/10.1186/s40164-024-00541-3. Additional file 1. Supplementary data includes supplementary material and methods, supplementary tables S1-2 and supplementary figure S1-S4 Author contributions EH, LM, AP, GT, and YZ performed study concept and design; EH and YZ performed writing; all authors provided acquisition, analysis and interpretation of data, EH performed statistical analysis; BP, ML, LM, and AP provided technical and materials support.EH, LM, and YZ contributed to review and revision.EH, VS, LM, AP, MT, AD, ML, BP, RX, CG, GT, and YZ read and approved the final paper. ", "section_name": "Supplementary Information", "section_num": null }, { "section_content": "This study was approved by the Institutional Review Board (JHIRB#00278530) and performed in accordance with the Declaration of Helsinki. ", "section_name": "Declarations Ethics approval and consent to participate", "section_num": null }, { "section_content": "A retrospective study and the informed consent was waived by the Institutional Review Board. ", "section_name": "Consent for publication", "section_num": null }, { "section_content": "The authors declare no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.1038/ncomms11938	Chromatin accessibility maps of chronic lymphocytic leukaemia identify subtype-specific epigenome signatures and transcription regulatory networks	<jats:title>Abstract</jats:title><jats:p>Chronic lymphocytic leukaemia (CLL) is characterized by substantial clinical heterogeneity, despite relatively few genetic alterations. To provide a basis for studying epigenome deregulation in CLL, here we present genome-wide chromatin accessibility maps for 88 CLL samples from 55 patients measured by the ATAC-seq assay. We also performed ChIPmentation and RNA-seq profiling for ten representative samples. Based on the resulting data set, we devised and applied a bioinformatic method that links chromatin profiles to clinical annotations. Our analysis identified sample-specific variation on top of a shared core of CLL regulatory regions. <jats:italic>IGHV</jats:italic> mutation status—which distinguishes the two major subtypes of CLL—was accurately predicted by the chromatin profiles and gene regulatory networks inferred for <jats:italic>IGHV</jats:italic>-mutated versus <jats:italic>IGHV</jats:italic>-unmutated samples identified characteristic differences between these two disease subtypes. In summary, we discovered widespread heterogeneity in the chromatin landscape of CLL, established a community resource for studying epigenome deregulation in leukaemia and demonstrated the feasibility of large-scale chromatin accessibility mapping in cancer cohorts and clinical research.</jats:p>	[ { "section_content": "hronic lymphocytic leukaemia (CLL) is the most common type of leukaemia in the Western world 1 .It is characterized by a remarkable clinical heterogeneity, with some patients pursuing an indolent course, whereas others progress rapidly and require early treatment.The diverse clinical course of CLL patients, in particular those that initially present with low disease burden, fuels interest in prognostic biomarkers and personalized therapies 2 .Current clinical biomarkers for CLL include mutational status of the IGHV genes 3,4 , IGHV gene family usage 5 , stereotyped B-cell receptors 6,7 , serum markers 8,9 , chromosomal aberrations 10,11 and somatic mutations [12][13][14] .Most notably, IGHV mutation status distinguishes between a less aggressive form of CLL with mutated IGHV genes (mCLL) and a more aggressive form with unmutated IGHV genes (uCLL).Several surrogate biomarkers of IGHV mutation status have been described.For example, high levels of ZAP70 expression appear to be associated with uCLL 15 .In addition to these focused biomarkers, transcriptome profiling has been used to define broader molecular signatures that may improve disease stratification independent of IGHV mutation status 16 . Recent genome and exome sequencing projects have identified additional genes that are recurrently mutated in CLL 17,18 , some of which have prognostic significance.Nevertheless, CLL samples carry relatively few genetic aberrations compared with other adult cancers 19 , and some patients develop progressive disease despite being classified as 'low risk' based on genetic markers, suggesting that non-genetic factors are relevant for CLL aetiology and outcome.Several lines of evidence point to a role of epigenome deregulation in CLL pathogenesis: first, somatic mutations have been observed in non-coding regions of the genome, where they appear to induce deregulation of relevant cancer genes 18 .Second, chromatin remodelling proteins such as ARID1A and CHD2 are recurrently mutated in CLL 17,18 , indicating causal links between chromatin deregulation and CLL.Third, aberrant DNA methylation was observed in all studied CLL patients [20][21][22] , correlated with IGHV mutation status and identified a new subtype (iCLL) that appears to be an intermediate between mCLL and uCLL 20,23 . Although prior studies of epigenome deregulation in primary cancer samples have focused almost exclusively on DNA methylation 24 , recent technological advances now make it possible to map chromatin landscapes in large patient cohorts.Most notably, the assay for transposase-accessible chromatin using sequencing (ATAC-seq) facilitates open chromatin mapping in scarce clinical samples 25 and ChIPmentation provides a streamlined, low-input workflow for genome-wide mapping of histone marks and transcription factors 26 .These two assays use a hyperactive variant of the prokaryotic Tn5 transposase, which integrates DNA sequencing adapters preferentially in genomic regions with accessible chromatin.ATAC-seq profiles are similar to those of DNase-seq, sharing the ability to detect footprints of transcription factor binding in the chromatin accessibility landscape 27 .ChIPmentation closely recapitulates the results obtained by more classical chromatin immunoprecipitation followed by sequencing protocols 26 .Both assays work well on scarce patient samples, and they enable fast sample processing on timescales that would be compatible with routine clinical diagnostics. To establish the feasibility of large-scale chromatin analysis in primary cancer samples and to provide a basis for dissecting regulatory heterogeneity in CLL, we performed chromatin accessibility mapping using the ATAC-seq assay on a cohort of 88 primary CLL samples derived from 55 patients.Furthermore, for ten of these samples we established histone profiles using ChIPmentation for three histone marks (H3K4me1, H3K27ac and H3K27me3) and transcriptome profiles using RNA sequencing (RNA-seq).We also developed a bioinformatic method for linking these chromatin profiles to clinical annotations and molecular diagnostics data, and we performed an initial analysis of gene regulatory networks that underlie the major disease subtypes of CLL.In summary, this study provides a publicly available reference data set and a rich source of testable hypotheses for dissecting CLL biology and pathogenesis. ", "section_name": "C", "section_num": null }, { "section_content": "Chromatin accessibility maps for 88 CLL samples.To map the chromatin accessibility landscape of CLL (Fig. 1a), we performed ATAC-seq on 88 purified lymphocyte samples obtained from the peripheral blood of 55 CLL patients.These patients were managed at a single medical centre, and they collectively represent the spectrum of clinical phenotypes that are commonly observed in CLL (Supplementary Data 1).Their average age at sample collection was 73 years, and 8% of patients were sampled at relapse following initial or subsequent therapy.The majority of samples (58%) had been classified as IGHV-mutated as part of routine clinical diagnostics (Supplementary Fig. 1 and Supplementary Data 1). All samples selected for ATAC-seq library preparation contained at least 80% leukaemic cells.The ATAC-seq libraries were sequenced with an average of 25.4 million fragments, resulting in a data set comprising a total of 2.2 billion sequenced fragments (Supplementary Data 2).Data quality was high in all cases, with mitochondrial read rates in the expected range for ATAC-seq (mean: 38.3%; s.d.: 9.3%) and the characteristic patterns of nucleosome phasing derived from paired-end data (Supplementary Fig. 2). The individual samples were sequenced with sufficient depth to recover the majority of chromatin-accessible regions that are detectable in each sample (Supplementary Fig. 3).Moreover, by combining data across all 88 samples we approached cohort-level saturation in terms of unique chromatin-accessible regions (Fig. 1b), indicating that our cohort is sufficiently large to identify most regulatory regions commonly accessible in CLL samples. As illustrated for the BLK gene locus (Fig. 1c), our ATAC-seq data set can be aggregated into a comprehensive map of chromatin accessibility in CLL.This map comprises 112,298 candidate regulatory regions, of which 11.6% are constitutively open across essentially all CLL samples, whereas 59.1% are open in a sizable proportion of samples (5-95% of samples) and 29.3% are unique to only one or very few samples (Supplementary Fig. 4a).All data are available for interactive browsing and download from the Supplementary Website (http://cll-chromatin.computational-epigenetics.org/). Chromatin-accessible regions in CLL are widely distributed throughout the genome, with moderate enrichment at genes and promoters (Fig. 1d and Supplementary Fig. 4b).We also compared the CLL-accessible regions with epigenome segmentations for CD19 þ B cells (Fig. 1e and Supplementary Fig. 4c), a related cell type for which comprehensive reference epigenome data are publicly available 28 .Strong enrichment was observed for regions that are classified as transcription start sites or as enhancer elements in the B cells, indicative of a globally similar chromatin accessibility landscape between B cells and CLL.Nevertheless, a sizable fraction of CLL-accessible regions carried quiescent or repressive chromatin in B cells, which is the expected pattern for regulatory elements that are subject to CLL-specific activation. Heterogeneity in the CLL chromatin accessibility landscape.Although the number of constitutively accessible regions in our cohort was relatively low (11.6%,Supplementary Fig. 4a), we still observed high consistency between individual samples and, any two samples in our data set shared 70-98% of their chromatinaccessible regions (Supplementary Fig. 5a).Conversely, we also observed robust differences in the ATAC-seq signal intensity between samples.To facilitate gene-by-gene investigation of this heterogeneity, we established the 'chromatin accessibility corridor' as a means of aggregating the cohort-level variation into a single intuitive genome browser track (Fig. 2a and Supplementary Website).As illustrated by the PAX5 and BCL6 gene loci, even where the locations of chromatin accessible regions are shared across most samples, substantial differences in the ATAC-seq intensity levels were observed (Fig. 2a). For a more systematic investigation of chromatin heterogeneity in CLL, we calculated the cohort-level variance for each of the 112,298 regions in the CLL consensus map and linked these regions to nearby genes that they may regulate (see Methods for details).Promoters of genes with a known role in B-cell biology and/or CLL pathogenesis showed significantly reduced variability (Po10 À 5 , Kolmogorov-Smirnov test; Supplementary Fig. 5b), which was not due to differential representation of CpG islands among the promoters of the gene sets (P ¼ 0.49, Fisher's exact test).For distal enhancer elements we did not observe any clear differences in heterogeneity between genes with and without a link to B cells and CLL (P ¼ 0.08, Kolmogorov-Smirnov test).Beyond these global trends, the variance and distribution of chromatin accessibility across samples was highly gene specific (Fig. 2b and Supplementary Fig. 5c), as illustrated by CLL-linked genes including B-cell surface markers (CD19), B-cell receptor signalling components (CD79A/B, LYN and BTK), common oncogenes (MYCN, KRAS and NRAS) and genes that are recurrently mutated in CLL (NOTCH1, SF3BP1, XPO1 and CDKN1B) 17,18,29 . Unsupervised principal component analysis clearly identified IGHV mutation status as the major source of heterogeneity in chromatin accessibility among CLL samples (Fig. 2c and Supplementary Fig. 6).However, the first two principal components explained only 6.8 and 5.2% of the total variance in the chromatin accessibility data set, suggesting that many other factors contribute to the observed differences between samples.The most direct way by which differences in chromatin accessibility may influence disease course would be through differential regulation of CLL-relevant genes.Therefore, to systematically assess the link between chromatin accessibility and gene expression in our cohort, we performed RNA-seq on ten CLL samples with matched ATAC-seq data.A weak positive correlation was observed between chromatin accessibility and gene expression (Pearson's r ¼ 0.33; Supplementary Fig. 7a), which was highly dependent on the distance of the chromatinaccessible region to the nearest transcription start site (Supplementary Fig. 7b). For chromatin-accessible regions in the vicinity of genes that RNA-seq identified as differentially expressed between IGHV-mutated (mCLL) and IGHV-unmutated (uCLL) samples (Supplementary Data 3), we observed significant differences in chromatin accessibility, which provided partial separation of the two disease subtypes (Supplementary Fig. 7c).A more pronounced separation was observed when we focused our .This cohort-level track uses colour-coded percentiles to visualize the observed heterogeneity between samples.The bottom row zooms in on the chromatin accessibility landscape at three specific regulatory regions.(b) Violin plots showing the cohort-wide distribution of chromatin accessibility at promoters (chromatin-accessible regions located within 2,500 bp from the transcription start site) and putative enhancers of genes with a known role in B-cell biology and/or CLL pathogenesis.(c) Unsupervised principal component analysis based on the chromatin accessibility for all 88 samples at each of the 112,298 chromatin-accessible regions in the CLL cohort.Samples are colour coded according to their IGHV mutation status, using o98% germline homology as threshold for classifying a sample as mutated. analysis on those regions that had been identified as differentially methylated between mCLL and uCLL in a prior study of DNA methylation in CLL 20 (Supplementary Fig. 7d).Finally, we assessed whether patterns of differential variability between mCLL and uCLL (that is, higher levels of heterogeneity in one or the other subtype) may provide insights into the biology of these two disease subtypes.We identified 389 regions that showed a higher degree of variability among mCLL samples, whereas 581 regions were more variable among uCLL samples (Supplementary Fig. 8a)-consistent with prior results showing higher gene expression variability among uCLL samples 30 .These differentially variable regions were distributed across a broad range of ATAC-seq intensity values and were not a side effect of differences in average chromatin accessibility (Supplementary Fig. 8b).Genomic region enrichment analysis using the LOLA software 31 found mCLL-variable regions enriched for B-cell-specific transcription factor binding (ATF2, BATF, BCL6, NFKB and RUNX3) and active histone marks (Supplementary Fig. 8c).In contrast, uCLL-variable regions were strongly associated with the cohesin complex, including binding sites for CTCF, RAD21 and SMC3. ", "section_name": "Results", "section_num": null }, { "section_content": "To link the CLL chromatin accessibility landscape to clinical annotations and molecular diagnostics data (most notably to the IGHV mutation status that distinguishes between mCLL and uCLL), we devised a machine learning-based method that derives subtype-specific signatures directly from the data (Fig. 3a).Random forest classifiers were trained to predict whether a sample is IGHV-mutated or IGHV-unmutated, based on the chromatin accessibility values for all 112,298 regions in the CLL consensus map.We evaluated the performance of the resulting classifier by leave-one-out cross-validation and observed excellent prediction accuracy with a receiver operating characteristic (ROC) area under curve of 0.96 (Fig. 3b), which corresponds to a sensitivity of 95.6% at a specificity of 88.2%.To confirm that this cross-validated test set performance was not inflated by any form of overtraining, we repeated the same predictions one thousand times with randomly shuffled class labels.Much lower ROC area under curve values were observed in all cases, and their mean was very close to the theoretical expectation of 0.5 (Fig. 3b). Next, we extracted the most predictive regions from the trained classifiers, giving rise to data-driven chromatin signatures that discriminate between mCLL and uCLL (Supplementary Data 4).Hierarchical clustering categorized these regions into 719 with increased chromatin accessibility in IGHV-mutated samples ('mCLL regions', cluster 1 in Fig. 3c) and 764 regions with increased chromatin accessibility in IGHV-unmutated samples ('uCLL regions', cluster 2 in Fig. 3c).More than half (51%) of these machine learning-based signature regions overlapped with statistically significant differential ATAC-seq peaks between IGHV-mutated and IGHV-unmutated samples (Supplementary Fig. 9a and Supplementary Data 4, see Methods for details).The remaining regions contributed to accurate prediction of CLL subtypes as part of a broader signature, even though they did not by themselves reach the stringent thresholds of the differential peak analysis (Supplementary Fig. 9b). To test whether these subtype-specific chromatin signatures reflected more general differences in the gene regulatory landscape of CLL, we compared RNA-seq profiles and ChIPmentation maps for three histone marks (H3K4me1, H3K27ac and H3K27me3) between five IGHV-mutated and five IGHV-unmutated samples.We found that the genes in the vicinity of the signature regions were on average more highly expressed in the cell type showing higher chromatin accessibility (Fig. 3d and Supplementary Fig. 10), although only a small percentage of these genes were significantly differentially expressed between mCLL and uCLL samples based on our RNA-seq data (0.8% and 6.3%, respectively).Moreover, the ChIPmentation profiles were consistently associated with the differences in chromatin accessibility.Higher levels of the active H3K27ac mark as compared with repressive H3K27me3 were found in mCLL samples and mCLL-specific regions, and vice versa for uCLL (Fig. 3e).This observation is illustrated by the ZNF667 promoter and an enhancer at the ZBTB20 locus (Fig. 3f), two genes that have been identified as predictors of time to treatment and overall survival in CLL 32,33 . Between individual samples we observed both qualitative (that is, the presence or absence of a peak) and quantitative (that is, different peak height) differences in chromatin accessibility, as illustrated by several genes with a known role in CLL (Supplementary Figs 11 and12).For example, the expression ratio between ADAM29 and LPL has been shown to have prognostic value in CLL 34 and our data set identifies an mCLL-specific chromatin-accessible region within the ADAM29 locus (Supplementary Fig. 11) as well as a uCLL-specific chromatin-accessible region overlapping with the LPL promoter (Supplementary Fig. 12), which may provide a regulatory basis for the previously described association.CD83, which has been associated with treatment-free survival 35 , is another example of a gene locus containing an mCLL-specific chromatin-accessible region (Supplementary Fig. 11).In contrast, uCLL-specific regions were identified in the gene loci encoding the CLL-linked transcription factor CREBBP 18 and the surface protein CD38, which has been extensively validated as a prognostic factor in CLL 36 (Supplementary Fig. 12). To gain insight into the more general biological characteristics of the mCLL and uCLL signature regions, we performed genomic region set analysis using LOLA 31 (Fig. 3g), and we observed that the mCLL regions were enriched for active promoter and enhancer regions (marked by H3K4me1 and H3K27ac) in lymphocyte-derived cell lines (SU-DHL-5, JVM-2, GM12878 and KARPAS-422), as well as binding sites of relevant transcription factors (BATF, BCL6 and BLC3).In contrast, the uCLL regions were enriched for H3K4me1-marked promoter/ enhancer regions in CD38-negative naive B cells, reflecting the postulated naive B-cell origin of these CLL cells 37 .The uCLL regions were also enriched for transcribed regions (H3K36me3) in naive B cells and in B-cell-derived cell lines such as the BL-2 cell line, which has not undergone class-switch recombination. We also performed motif enrichment analysis for the mCLL and uCLL signature region sets and, we observed significant enrichment relative to a random background model but no clear-cut differences when comparing the two region sets directly with each other (which is expected given the low statistical power of such an analysis).Nevertheless, when we linked chromatin-accessible regions to co-localized genes, we observed strong differences in the enrichment for cellular signalling pathways (Fig. 3h).The mCLL regions were associated with pathways having an established role in normal lymphocytes (CTLA4 inhibitory signaling, high-affinity IgE receptor signalling, Fc epsilon signalling and Fc gamma receptor signalling), whereas the uCLL regions were associated with cancer-associated pathways such as NOTCH signalling and fibroblast growth factor receptor signalling.All of these enrichment analyses were validated based on the statistically significant differential ATAC-seq peaks between IGHV-mutated and IGHV-unmutated samples, which gave rise to highly similar results (Supplementary Fig. 13). Finally, we investigated whether a third CLL subtype-termed IGHV intermediate (iCLL)-could be detected in our data set, as it was recently proposed based on DNA methylation data 20,23 .Clustering all samples based on the IGHV mutation signature regions, we indeed observed two intermediate clusters, the larger one comprising 20 samples from 14 patients (Fig. 4a, green) and the smaller one comprising 3 samples from 2 patients (Fig. 4a, brown).Most but not all of these iCLL samples were classified as IGHV-mutated based on the available molecular diagnostics data (Supplementary Fig. 14).Principal component analysis provided further evidence that the iCLL samples fall between mCLL and uCLL samples based on their ATAC-seq profiles (Fig. 4b).Their intermediate character was also supported by the RNA-seq and ChIPmentation data, where the iCLL group showed patterns that consistently ranged between those of the mCLL and uCLL groups (Supplementary Fig. 15). Gene regulatory networks in mCLL and uCLL disease subtypes. In addition to providing chromatin accessibility maps, ATAC-seq can also detect transcription factor binding based on characteristic chromatin footprints 25 .Using this property of our data, we inferred chromatin-based gene regulatory networks for CLL and its two major disease subtypes (Fig. 5a).To that end, we pooled all ATAC-seq data across the analysed samples, identified footprints for 366 transcription factors with high-quality motifs in the JASPAR database 38 and linked these regulatory elements to their putative target genes (see Methods for details).The quality of the observed footprints was comparable to those in publicly available DNase-seq data for CD19 þ B cells (Supplementary Fig. 16), although we observed some deviations between the two assays that are likely due to the different sequence specificity of the Tn5 enzyme as opposed to the DNase I enzyme. We first inferred a pan-CLL gene regulatory network using ATAC-seq data from all samples (Supplementary Fig. 17).The resulting network was dominated by highly connected transcription factors, including broadly activating factors (SP1/2/3), the insulator protein CTCF and regulators of biological processes such as cell proliferation (EGR), cell cycle (E2F) and B-cell maturation (SPI1 and PAX5).This pan-CLL network was structurally similar to a network for CD19 þ B cells that we inferred from publicly available DNase-seq data using the same bioinformatic method (Supplementary Fig. 18), and in the absence of a large chromatin accessibility data set of B cells from healthy individuals it is not possible to conclusively identify the CLL-specific parts of our network. Second, to investigate regulatory differences between CLL subtypes, we inferred gene regulatory networks separately for mCLL and uCLL samples (Supplementary Fig. 19) and identified the most differentially connected genes between the two (Fig. 5b). Genes that were more highly connected in the mCLL network coded for the transcription factors ZNF354C and ELF5, the metallopeptidase ADAM29 and the membrane protein CD22.In contrast, the BMP receptor CRIM1, the transcription factors MECOM and PAX9, the fibroblast growth factor signalling receptor FGFR1 and the membrane protein CD9 were more highly connected in the uCLL network (Fig. 5c).The more highly connected genes in either subtype also showed higher levels of H3K4me1 and H3K27ac in their regulatory elements in samples of the corresponding subtype (Supplementary Fig. 20a,b). When we restricted our analysis to genes with a known role in B-cell biology and/or CLL pathogenesis (Fig. 5d), we observed a highly specific association of CD22 (which codes for an inhibitory receptor involved in B-cell receptor signalling) with mCLL, whereas CD38 and ZAP70 were preferentially associated with uCLL.Focusing on CD22 and PAX9 as two high-ranking genes in our analysis, we plotted the sub-networks of their direct neighbours and observed characteristic differences between the gene regulatory networks for mCLL and uCLL (Supplementary Fig. 20c).Many of the subtype-specific genes identified by the regulatory network also showed locus-specific differences in their ChIPmentation profiles (Supplementary Fig. 20d).Altogether, our results confirm that ATAC-seq profiles are useful for identifying epigenome differences in clinical samples, and they illustrate how this data set can be used for deriving testable hypotheses about the regulatory basis of CLL. ", "section_name": "Disease subtype-specific patterns of chromatin accessibility.", "section_num": null }, { "section_content": "By ATAC-seq profiling on a large set of primary CLL samples, we have established a detailed map of the chromatin accessibility landscape in CLL.The ATAC-seq data were complemented by RNA-seq profiles and ChIPmentation for three histone marks, performed in ten representative samples covering three disease subtypes (mCLL, uCLL and iCLL).To our knowledge, this data set is currently the largest catalogue of chromatin accessibility maps for any cancer type, demonstrating the feasibility of chromatin profiling in large cohorts of primary cancer samples and validating a broadly applicable bioinformatics workflow for analysing such data. The large number of patient samples included in this study allowed us to dissect the role of epigenome variability as a potential contributor to cancer heterogeneity 39 .We found that variability between samples was common in our data set, both in the form of qualitative (that is, the presence or absence of a peak) and quantitative (that is, different peak height) differences between individual samples.In the absence of a reference data set with chromatin accessibility maps for normal B cells from a large number of healthy donors, it remains unclear whether or not the observed heterogeneity in CLL constitutes a major increase over the expected heterogeneity in a genetically diverse cohort.Nevertheless, significantly reduced heterogeneity at the promoters of genes involved in B-cell biology and/or CLL pathogenesis suggest a functional role of the observed interindividual differences.Overall, our data support the existence of a core regulatory landscape shared by most or all CLL samples, which is complemented by sample-specific subsets of a substantially larger number of CLL-associated regulatory regions. IGHV mutation status was the single biggest contributor to sample-specific differences in chromatin accessibility, although it explained only 5-10% of the observed variance in our data set.Based on the ATAC-seq profiles we were able to distinguish with excellent accuracy between IGHV-mutated mCLL and IGHV-unmutated uCLL.Our analysis also suggested the existence of one (or possibly two) intermediate type (iCLL), consistent with a recent report that used DNA methylation analysis of a large CLL cohort to identify novel CLL subtypes 20 .Chromatin accessibility and DNA methylation both appear to separate better between these disease subtypes than gene expression data, suggesting that the biological differences between the major subtypes of CLL are primarily encoded in the epigenome and possibly reflect patterns retained from a subtype-specific cell-of-origin. Combining data across samples provided sufficient sequencing depth for footprinting analysis of transcription factor binding, allowing us to infer gene regulatory networks from the data and to compare them between mCLL and uCLL.Although genomic footprinting has its limitations 40 , the resulting network models give rise to predictions that can provide a starting point for further experimental dissection of the transcription regulatory landscape of CLL.For example, mCLL-associated regions were enriched for transcription factors that are active in mature B cells and involved in memory B-cell differentiation (BATF and BCL6), whereas the uCLL group was enriched for regulatory regions that are active in other haematopoietic cell types, indicative of a less differentiated cell state.Moreover, pathways that may boost proliferation, such as NOTCH signalling 41 and interferon signalling 42 , were specifically observed in the more aggressive subtype (uCLL), whereas enrichment of inhibitory signalling by CTLA4 may contribute to the more indolent character of mCLL 43 .Beyond a small number of specific differences, the inferred gene regulatory networks were highly similar between mCLL and uCLL, consistent with the low number of differentially expressed genes that were previously observed between CLL subtypes 16,44,45 . From a technological perspective, our study describes broadly applicable methods for dissecting chromatin profiles in large cohorts of primary patient samples.The differential chromatin analysis outlined in Fig. 3 starts from clinical and/or diagnostic data and uses supervised learning techniques to identify and cross-validate discriminatory chromatin signatures.We focused specifically on IGHV mutation status, but the method can be applied to any type of patient grouping, for example, based on disease progression or therapy response.Moreover, the described method for ATAC-seq-based inference of gene regulatory networks (Fig. 5) establishes a data-driven approach for dissecting regulatory cell states-including their differences between disease subtypes-that is highly complementary to previous work aimed at inferring regulatory networks from transcriptome data [46][47][48] .Finally, the 'chromatin accessibility corridor' (Fig. 2) adapts a related concept 49 to provide intuitive browser-based visualization of chromatin data across large cohorts, while accounting for regulatory heterogeneity. Relevant limitations of our study include the following: (i) lack of a clearly defined and experimentally accessible cell-of-origin for uCLL and mCLL, making it difficult to distinguish with certainty between chromatin patterns that are CLL specific and those that are derived from the disease's cell-of-origin; (ii) clonal heterogeneity of CLL within patients, which would be experimentally addressable only with single-cell sequencing technologies 50,51 that are currently limited in their genomewide coverage; (iii) lack of scalable methods for distinguishing between functional and non-functional transcription factor binding; and (iv) ambiguities in the assignment of transcription factor binding sites to the genes that they regulate.In the light of these limitations, the inferred gene regulatory networks constitute an initial model that will require future refinement as additional data and validations become available. In summary, our study establishes a chromatin accessibility landscape of CLL, which identifies shared gene regulatory networks as well as widespread heterogeneity between individual patients and between disease subtypes.It also provides a resource that can act as a starting point for deeper dissection of chromatin regulation in CLL, identification of therapeutically relevant mechanisms and eventual translation of relevant discoveries into clinical practice.Given that the chromatin profiling assays used here (ATAC-seq and ChIPmentation) are sufficiently fast and straightforward for use in a clinical sequencing laboratory, chromatin deregulation is becoming increasingly tractable as a promising source of biomarkers for stratified cancer therapy. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Sample acquisition and clinical data.All patients were diagnosed and treated at the Royal Bournemouth Hospital (UK) according to the revised guidelines of the International Workshop Chronic Lymphocytic Leukemia/National Cancer Institute.Patients were selected to reflect the clinical and biological heterogeneity of the disease.Sequential samples were included for a total of 24 patients.All samples contained more than 80% leukaemic cells.Established chromosomal rearrangements were diagnosed by fluorescence in situ hybridization (Abbott Diagnostics; DakoCytomation) or multiple ligation-dependent probe amplification using the MLPA P037 CLL-1 probemix (MRC Holland SALSA) according to the manufacturers' instructions.Chromosome analysis was performed and reported according to the International System for Human Cytogenetic Nomenclature.IGHV was sequenced as previously described 4 , and a threshold of 498% germline homology was taken to define the unmutated subset 4 .The study was approved by the ethics committees of the contributing institutions (Royal Bournemouth Hospital and Medical University of Vienna).Informed consent was obtained from all participants.ATAC sequencing.Accessible chromatin mapping was performed using the ATAC-seq method as previously described 25 , with minor adaptations.In each experiment, 10 5 cells were washed once in 50 ml PBS, resuspended in 50 ml ATAC-seq lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl 2 and 0.1% IGEPAL CA-630) and centrifuged for 10 min at 4 °C.On centrifugation, the pellet was washed briefly in 50 ml MgCl 2 buffer (10 mM Tris pH 8.0 and 5 mM MgCl 2 ) before incubating in the transposase reaction mix (12.5 ml 2 Â TD buffer, 2 ml transposase (Illumina) and 10.5 ml nuclease-free water) for 30 min at 37 °C.After DNA purification with the MinElute kit, 1 ml of the eluted DNA was used in a quantitative PCR (qPCR) reaction to estimate the optimum number of amplification cycles.Library amplification was followed by SPRI size selection to exclude fragments larger than 1,200 bp.DNA concentration was measured with a Qubit fluorometer (Life Technologies).Library amplification was performed using custom Nextera primers 25 .The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq3000/4000 platform and the 25-bp paired-end configuration. RNA sequencing.Total RNA was isolated using the AllPrep DNA/RNA Mini Kit (Qiagen).RNA amount was measured using Qubit 2.0 Fluorometric Quantitation (Life Technologies), and the RNA integrity number was determined using Experion Automated Electrophoresis System (Bio-Rad).RNA-seq libraries were prepared using a Sciclone NGS Workstation (PerkinElmer) and a Zepyhr NGS Workstation (Perki-nElmer) with the TruSeq Stranded mRNA LT sample preparation kit (Illumina).Library amount and quality were determined using Qubit 2.0 Fluorometric Quantitation (Life Technologies) and Experion Automated Electrophoresis System (Bio-Rad).The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-read configuration. ChIPmentation.ChIPmentation was carried out as previously described 26 , with minor adaptions.Briefly, cells were washed once with PBS and fixed with 1% paraformaldehyde in up to 1 ml PBS for 10 min at room temperature.Glycine was added to stop the reaction.Cells were collected at 500 g for 10 min at 4 °C (subsequent work was performed on ice and used cool buffers and solutions unless otherwise specified) and washed twice with up to 0.5 ml ice-cold PBS supplemented with 1 mM phenylmethyl sulfonyl fluoride (PMSF).The pellet was lysed in sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.25% SDS, 1 Â protease inhibitors (Sigma) and 1 mM PMSF) and sonicated with a Covaris S220 sonicator for 20-30 min in a milliTUBE or microTUBE until the size of most fragments was in the range of 200-700 bp.Lysates were centrifuged at full speed for 5 min at 4 °C, and the supernatant containing the sonicated chromatin was transferred to a new tube.The lysate was then brought to RIPA buffer conditions (final concentration: 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1 Â protease inhibitors (Sigma) and 1 mM PMSF) to a volume of 200 ml per immunoprecipitation.For each immunoprecipitation, 10 ml magnetic Protein A (Life Technologies) were washed twice and resuspended in PBS supplemented with 0.1% BSA.The antibody was added and bound to the beads by rotating 2 h at 4 °C.Used antibodies were H3K4me1 (0.5 mg per immunoprecipitation, Diagenode pAb-194-050), H3K27ac (1 mg per immunoprecipitation, Diagenode pAB-196-050) and H3K27me3 (1 mg per immunoprecipitation, Millipore 07-499).For control libraries, an immunoprecipitation with 2.5 mg of a nonspecific IgG rabbit antibody was used.Blocked antibody-conjugated beads were then placed on a magnet, supernatant was removed and the sonicated lysate was added to the beads followed by incubation for 3-4 h at 4 °C on a rotator.Beads were washed subsequently with RIPA (twice), RIPA-500 (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.1% DOC) (twice) and RIPA-LiCl (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 1% Triton X-100, 0.5% DOC and 0.5% NP40) (twice). Beads were washed once with cold Tris-Cl pH 8.0, to remove detergent, salts and EDTA.Beads were washed once more with cold Tris-Cl pH 8.0 but the reaction was not placed on a magnet to discard supernatant immediately.Instead, the whole reaction including beads was transferred to a new tube and then placed on a magnet to remove supernatant to decrease background.Beads were then carefully resuspended in 25 ml of the tagmentation reaction mix (10 mM Tris pH 8.0, 5 mM MgCl 2 , 10% v/v dimethylformamide) containing 1 ml Tagment DNA Enzyme from the Nextera DNA Sample Prep Kit (Illumina) and incubated at 37 °C for 1-3 min in a thermocycler.The beads were washed with RIPA (twice) and once with cold Tris-Cl pH 8. Beads were washed once more with cold Tris-Cl pH 8.0 but the reaction was not placed on a magnet to discard supernatant immediately.Instead, the whole reaction including beads was again transferred to a new tube and then placed on a magnet to remove supernatant.Beads were then incubated with 70 ml elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA and 10 mM Tris-HCl pH 8.0) containing 2 ml of Proteinase K (NEB) for 1 h at 55 °C and 8 h at 65 °C, to revert formaldehyde cross-linking, and supernatant was transferred to a new tube.Finally, DNA was purified with SPRI AMPure XP beads (sample-to-beads ratio 1:2) or Qiagen MinElute columns. One microlitre of each library was amplified in a 10-ml qPCR reaction containing 0.15 mM primers, 1 Â SYBR Green and 5 ml Kapa HiFi HotStart ReadyMix (Kapa Biosystems), to estimate the optimum number of enrichment cycles with the following programme: 72 °C for 5 min, 98 °C for 30 s, 24 cycles of 98 °C for 10 s, 63 °C for 30 s and 72 °C for 30 s, and a final elongation at 72 °C for 1 min.Kapa HiFi HotStart ReadyMix was incubated at 98 °C for 45 s before preparation of all PCR reactions (qPCR and final enrichment PCR), to activate the hot-start enzyme for successful nick translation at 72 °C in the first PCR step.Final enrichment of the libraries was performed in a 50-ml reaction using 0.75 mM primers and 25 ml Kapa HiFi HotStart ReadyMix.Libraries were amplified for N þ 1 cycles, where N is equal to the rounded-up Cq value determined in the qPCR reaction.Enriched libraries were purified using SPRI AMPure XP beads at a beads-to-sample ratio of 1:1, followed by a size selection using AMPure XP beads to recover libraries with a fragment length of 200-400 bp.Library preparation was performed using custom Nextera primers as described for ATAC-seq 25 .The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq3000/4000 platform and the 25-bp paired-end configuration. Preprocessing of the ATAC-seq data.Reads were trimmed using Skewer 52 .Trimmed reads were aligned to the GRCh37/hg19 assembly of the human genome using Bowtie2 (ref.53) with the '-very-sensitive' parameter.Duplicate reads were removed using sambamba markdup 54 , and only properly paired reads with mapping quality 430 and alignment to the nuclear genome were kept.All downstream analyses were performed on the filtered reads.Genome browser tracks were created with the genomeCoverageBed command in BEDTools 55 and normalized such that each value represents the read count per base pair per million mapped and filtered reads.Finally, the UCSC Genome Browser's bedGraphToBigWig tool was used to produce a bigWig file.Combined tracks with percentile signal across the cohort were created by quantifying ATAC-seq read coverage at every reference genome position using BEDTools coverage and normalizing it between samples.Normalization was done by dividing each value by the total number of filtered reads and multiplying it with ten million, to obtain numbers that are comparable and easy to visualize.Next, the mean as well as the 5th, 25th, 75th and 95th percentiles of signal across the whole cohort were calculated with Numpy, converted into bedgraph files and subsequently to bigwig format using bedGraphToBigWig.Peak calling was performed with MACS2 (ref.56) using the '-nomodel' and '-extsize 147' parameters, and peaks overlapping blacklisted features as defined by the ENCODE project 57 were discarded. Preprocessing of the RNA-seq data.Reads were trimmed with Trimmomatic 58 and aligned to the GRCh37/hg19 assembly of the human genome using Bowtie1 (ref.59) with the following parameters: -q -p 6 -a -m 100-minins 0-maxins 5000-fr-sam-chunkmbs 200.Duplicate reads were removed with Picard's MarkDuplicates utility with standard parameters before transcript quantification with BitSeq 60 using the Markov chain Monte Carlo method and standard parameters.To obtain gene-level quantifications, we assigned the expression values of its highest expressed transcript to each gene.Differential gene-level expression between the three IGHV mutation status groups was performed using DESeq2 (ref.61) from the raw count data with a significance threshold of 0.05.To produce genome browser tracks, we mapped the reads to the genomic sequence of the GRCh37/hg19 assembly of the human genome using Bowtie2 (ref.53) with the '-very-sensitive' parameter, removed duplicates using sambamba markdup 54 and used the genomeCoverageBed command in BEDTools 55 to produce a bedgraph file.This file was normalized such that each value represents the read count per base pair per million filtered reads, and the UCSC Genome Browser's bedGraphToBigWig tool was used to convert it into a bigWig file. Preprocessing of the ChIPmentation data.Reads were trimmed using Skewer 52 .Trimmed reads were aligned to the GRCh37/hg19 assembly of the human genome using Bowtie2 (ref.53) with the '-very-sensitive' parameter.Duplicate reads were removed using sambamba markdup 54 , and only properly paired reads with mapping quality 430 and alignment to the nuclear genome were kept.All downstream analyses were performed on the filtered reads.Genome browser tracks were created with the genomeCoverageBed command in BEDTools 55 and normalized such that each value represents the read count per base pair per million filtered reads.Finally, the UCSC Genome Browser's bedGraphToBigWig tool was used to produce a bigWig file. Bioinformatic analysis of chromatin accessibility.The CLL consensus map was created by merging the ATAC-seq peaks from all samples using the BEDTools 55 merge command.To produce Fig. 1b, we counted the number of unique chromatin-accessible regions after merging peaks for each sample in an iterative manner, randomizing the sample order 1,000 times and computing 95% confidence intervals across all iterations.The chromatin accessibility of each region in each sample was quantified using Pysam, counting the number of reads from the filtered BAM file that overlapped each region.To normalize read counts across samples, we performed quantile normalization using the normalize.quantilesfunction from the preprocessCore package in R. For each genomic region we calculated the support as the percentage of samples with a called peak in the region, and we calculated four measures of ATAC-seq signal variation across the cohort: mean signal, s.d., variance-to-mean ratio and the squared coefficient of variation (the square of the s.d.over the mean).In addition, we used BEDTools intersect to annotate each region with the identity of and distance to the nearest transcription start site and the overlap with Ensembl gene annotations (promoters were defined as the 2,500-bp region upstream of the transcription start site).Annotation with chromatin states was based on the 15-state genome segmentation for CD19 þ B cells from the Roadmap Epigenomics Project 62 (identifier: E032). To summarize the chromatin accessibility signals into one value per gene (Fig. 2b and Supplementary Fig. 5), we used the accessibility values of the closest region (but no further than 1,000 bp from the transcription start site) to represent the promoter and the mean values of all distal regions (located more than 2,500 bp from the transcription start site) of each gene to represent distal regulatory elements.To test for overrepresentation of CpG islands in the promoters of genes with a known role in B-cell biology and/or CLL pathogenesis, we downloaded the position of CpG islands in the GRCh37/hg19 assembly from the UCSC Genome Browser 63 , counted the number of promoters (as defined above) that overlapped by at least 1 bp with CpG islands in the gene set of interest and in all other genes with accessible elements in CLL, and used Fisher's exact test to assess the significance of the association.Unsupervised principal component analysis was performed with the scikit-learn 64 library (sklearn.decomposition.PCA) applied to the chromatin accessibility values of all chromatin-accessible regions across the CLL cohort. To investigate variability within the mCLL and uCLL sample groups, we divided the samples in two groups based on their IGHV mutation status (samples below a 98% homology threshold were considered mutated, and samples with missing values for the IGHV mutation status were excluded from the analysis) and we used the F test from the var.test function in R on the chromatin accessibility values of all CLL cohort regions.Significantly variable regions were defined as having a Bonferroni-corrected P-value below 0.05 and mean accessibility above 1.Region set-enrichment analysis was performed on the significantly variable regions of each group using LOLA 31 with its core databases: transcription factor binding sites from ENCODE 57 , tissue clustered DNase hypersensitive sites 65 , the CODEX database 66 , UCSC Genome Browser annotation tracks 63 , the Cistrome database 67 and data from the BLUEPRINT project 68 .Motif enrichment analysis was performed with the AME tool from the MEME suite 69 using 250 bp sequences centred on the chromatin-accessible regions and randomly generated sequences of the same length and set size from a distribution of zeroth-and first-order Markov order (single nucleotides and dinucleotide) frequencies as background. Machine learning analysis of disease subtypes.Random forest classifiers from the scikit-learn 64 Python library (sklearn.ensemble.RandomForestClassifier) were trained with the samples' IGHV mutation status as class label and the chromatin accessibility values for each sample at each of the 112,298 consensus regions as input features (prediction attributes).All samples with known IGHV mutation status were used for class prediction, the performance was evaluated by leave-oneout cross-validation, and the results were plotted as ROC curves using scikit-learn.Given that several patients contributed more than one sample to the cohort, in each iteration of the cross-validation we removed any samples from the training set that belonged to the same patient as the sample in the test set, to eliminate a potential risk of overtraining.Furthermore, we repeated the cross-validation 1,000 times based on randomly shuffled class labels to confirm that no overtraining occurred in our analysis.The most predictive regions for IGHV mutation status were selected by averaging the feature importance of the random forest classifiers over all iterations of the cross-validation and selecting those features with Gini importance higher than 10 À 4 .Region set enrichment was performed using LOLA 31 as described above.Pathway enrichment analysis was performed using seq2pathway 70 .The sample clustering in Fig. 4a was based on the pairwise correlation of ATAC-seq signal in the predictive regions between samples, and the dendrogram was plotted using Scipy's hierarchical clustering function.With the same values of chromatin accessibility from above, we performed principal component analysis on the CLL samples using R's implementation in the prcomp function.To provide further validation of the machine learning analysis, we also identified differential ATAC-seq peaks between IGHV-mutated and IGHV-unmutated samples using the DESeq2 R package 61 .This statistical analysis was based on read counts for all CLL-accessible regions in each patient, testing for differential chromatin accessibility using a model based on the negative binomial distribution.Regions with Benjamini-Hochberg adjusted P-values below 0.01 and an absolute log2 fold change above 1 were used for comparison with those signature regions identified by the machine-learning analysis. Gene regulatory network inference.Transcription factor binding maps as the basis for inferring gene regulatory networks were derived by footprinting analysis using the PIQ software 71 and a set of 366 human transcription factor motifs from the JASPAR database 38 .We retained only those transcription factors with at least 500 high-purity (40.7) binding sites overlapping with an ATAC-seq peak, as previously described 72 .Binding sites located in the gene body or in the 2,500-bp region upstream of its transcription start site were assigned to the overlapping gene(s), and intergenic binding sites were assigned to the gene whose transcription start site was closest to the peak.This assignment was based on the Ensembl gene annotation version 75, and we treated non-protein-coding genes in the same way as protein-coding genes.To infer gene regulatory networks, an interaction score was calculated in a similar way as previously described 72 : the interaction score between a transcription factor t and a gene g (S t,g ) was defined as the sum over all n transcription factor binding sites of t that can be assigned to g: S t;g ¼ X n i¼1 2Ã P i À 0:5 ð Þ Ã 10 À d i;g 100000 À Á In this formula P i is the PIQ purity score, and d i,g is the distance of a particular transcription factor binding site i to gene g.This score establishes a unidirectional (transcription factors to genes) and weighted (based on the interaction score) relationship, providing the edges of the gene regulatory network.We inferred gene regulatory networks for all samples combined and also separately for the two disease subtypes (mCLL and uCLL) based on IGHV mutation status.We considered only transcription-factor-to-gene interactions with scores above 1, and in Fig. 5b and Supplementary Figs 17 and 19 we plotted only nodes with more than 200 connections.For the CD19 þ B-cell gene regulatory network we used DNaseseq data from the Roadmap Epigenomics Project 62 (identifier: E032).Both the processing of the raw data and the network inference were performed in the same manner as for ATAC-seq.The comparison of composition and structural characteristics of the gene regulatory networks inferred from ATAC-seq data for the CLL cohort and from DNase-seq data for CD19 þ B cells was done using functions from the networkx 73 library in Python.The inferred networks were visualized using the Gephi software, applying the Force Atlas 2 graph layout with LinLog and hub dissuasion.To compare the inferred mCLL and uCLL networks, we divided the degree of each node by the total number of edges in each network, which compensates for differences in the absolute number of detected interactions, and quantified differences by subtracting and log2-transforming this value between networks for each node. ", "section_name": "Methods", "section_num": null } ]	[ { "section_content": "We thank all patients who have donated their samples for this study.We also thank Amelie Kuchler and Thomas Penz for expert technical assistance, Johanna Klughammer and Nathan Sheffield for their contributions to the data analysis pipeline, the Biomedical Sequencing Facility at CeMM for assistance with next-generation sequencing and all members of the Bock lab for their help and advice.Moreover, we thank Ulrich Ja ¨ger, Medhat Shehata, Philipp Staber and Jo ¨rg Menche for their comments and for critically reading the manuscript.This work was performed in the context of the BLUEPRINT project (European Union's Seventh Framework Programme grant agreement number 282510) and the ERA-NET projects EpiMark (FWF grant agreement number I 1575-B19) and CINOCA (FWF grant agreement number I 1626-B22).C.S. was supported by a Feodor Lynen Fellowship of the Alexander von Humboldt Foundation.J.C.S. was supported by Bloodwise (11052 and 12036), the Kay Kendall Leukaemia Fund (873), Cancer Research UK (C34999/A18087 and ECMC C24563/A15581), Wessex Medical Research and the Bournemouth Leukaemia Fund.C.B. was supported by a New Frontiers Group award of the Austrian Academy of Sciences. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Data availability.All data are available as genome browser tracks for interactive browsing and download from the Supplementary Website (http://cll-chromatin.computational-epigenetics.org/).The processed data are also openly available from NCBI GEO under the accession number GSE81274, whereas the raw sequencing data are available from EBI EGA under the accession number EGAS00001001821, under a controlled access regimen to protect the privacy of the patients who have donated the samples. ", "section_name": "", "section_num": "" }, { "section_content": "Accession codes: The processed high-throughput sequencing data are openly available from NCBI GEO under the accession number GSE81274, whereas the raw sequencing data are available from EBI EGA under the accession number EGAS00001001821, under a controlled access regimen to protect the privacy of the patients who have donated the samples.Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications Competing financial interests: The authors declare no competing financial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/ How to cite this article: Rendeiro, A. F. et al.Chromatin accessibility maps of chronic lymphocytic leukaemia identify subtype-specific epigenome signatures and transcription regulatory networks.Nat.Commun.7:11938 doi: 10.1038/ncomms11938 (2016).This work is licensed under a Creative Commons Attribution 4.0 International License.The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ ", "section_name": "Author contributions", "section_num": null }, { "section_content": "", "section_name": "Author contributions", "section_num": null }, { "section_content": "Accession codes: The processed high-throughput sequencing data are openly available from NCBI GEO under the accession number GSE81274, whereas the raw sequencing data are available from EBI EGA under the accession number EGAS00001001821, under a controlled access regimen to protect the privacy of the patients who have donated the samples.Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications Competing financial interests: The authors declare no competing financial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/ How to cite this article: Rendeiro, A. F. et al.Chromatin accessibility maps of chronic lymphocytic leukaemia identify subtype-specific epigenome signatures and transcription regulatory networks.Nat.Commun.7:11938 doi: 10.1038/ncomms11938 (2016).This work is licensed under a Creative Commons Attribution 4.0 International License.The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ ", "section_name": "Additional information", "section_num": null } ]
10.3324/haematol.10873	Chimeric T-cell receptors: new challenges for targeted immunotherapy in hematologic malignancies	Chimeric T-cell receptors (ChTCR), are a fascinating technological step in the field of immunotherapy for orienting the activity of immune cells towards specific molecular targets expressed on the cell surface of various tumors, including hematologic malignancies. The main characteristics of ChTCR are their ability to redirect T-cell specificity and their killing/effector activity toward a selected target in a non MHC-restricted manner, exploiting the antigen binding properties of monoclonal antibodies. ChTCR are, in fact, artificial T-cell receptors constituted by an antigen-recognizing antibody molecule linked to a T-cell triggering domain. Various hematologic malignancies represent optimal targets for the exploitation of ChTCR, because of the bright expression of specific antigens on the surface of tumor cells. Thus, CD19 and CD20 have been targeted for B-cell lymphoid tumors (acute lymphoblastic leukemia-ALL, lymphomas and chronic lymphocytic leukemia-CLL), CD33 for myeloid leukemia, and CD30 for lymphomas. Even though technical and safety progresses are still needed to improve the profile of gene transfer and protein expression of ChTCR, phase 1 trials will be carried out in the near future to demonstrate the feasibility of their clinical translation and, it is be hoped, give preliminary indications about their anti-tumor efficacy.	[ { "section_content": "][3][4] Comprehension of the mechanisms of tumor escape represents one of the most relevant acquisitions in immunology and has opened a new challenge for scientists and physicians. 5,6Tumor cells, in fact, evade recognition and elimination by immune effectors in many ways: i) low or absent expression of tumor-specific antigens; ii) expression of antigens that are shared with normal cells at certain developmental stages, so that the immune sys-tem has become self-tolerant or anergic; iii) down-regulation of surface expression of MHC molecules; iv) defective pathways of antigen processing and presentation; v) absence of appropriate co-stimulation to deliver a complete activation stimulus to effector T cells; vi) the presence of inhibitory molecules actively secreted by the tumor itself or by the tumor microenvironment (such as interleukin-10 and transforming growth factor-β); 7 and vii) the expansion of naturally occurring or tumorinduced T cells with regulatory activity. 8,9esides being capable of efficiently overcoming these multiple mechanisms of immune escape, any application of immunotherapy must consider all the practical issues related to the production of immune cells for clinical use: i) the choice of the right antigen(s) to ensure use in a sufficiently wide category of patients; ii) the development of methods of manipulation, which should be consistently straightforward and not excessively timeconsuming, in order to obtain large numbers of cells in a suitable framework; iii) the use of appropriate Good Manufacturing Practice (GMP)-grade reagents and procedures which guarantee the exploitation of rigid rules of cell manipulation; 10 iv) the consideration of safety issues related to the possible occurrence of genetic aberrations when using vectors for gene delivery. 113][14][15][16] The main characteristics of ChTCR are their ability to redirect T-cell specificity and their killing/effector activity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies.ChTCR are, in fact, artificial Tcell receptors constituted by an antigen-recognizing antibody molecule linked to a T-cell triggering domain. The scope of this review is not to describe the technical details concerning the construction of these artificial molecules (reviewed elsewhere 13 ), but to illustrate their functional immune aspects and their current or potential impacts on immunotherapy for hematologic malignancies. ", "section_name": "E", "section_num": null }, { "section_content": "8][19][20] Unfortunately, in the context of tumors, the specific antigens are often weak, poorly expressed on the cell surface, or presented in an inappropriate or incomplete way, often accompanied by secretion of inhibitory T-cell factors.Moreover, most tumors can elude major histocompatibility complex (MHC)-restricted T-cell-mediated immune recognition. 5,6For these reasons, enrichment and expansion of CTL targeting tumor-associated antigens are time-consuming and often ineffective, due to the low frequency of tumor-specific precursors in vivo. 163][14][15][16] Several artificial ChTCR have been devised in the last decade. 21These molecules are con-structed to express a specific antigen-binding domain (the extracellular domain, consisting of the variable chains of a monoclonal antibody), linked together as a single chain Fv (scFv), and a signaling region (the intracellular domain), usually taken from the zeta-chain of the TCR/CD3 complex (Figure 1).When expressed by T cells, the chimeric receptor links up the targeted antigen and triggers the cytolytic cascade of T cells, thus consecutively inducing the killing of the target population. 22,23he main advantage of this approach is that it relies on the construction of a universal receptor towards a selected single molecule, whose recognition is non-MHC-restricted and independent of antigen processing, thus bypassing all major mechanisms of tumor escape. ", "section_name": "Use of CHTCR-expressing T cells for tumor immunotherapy", "section_num": null }, { "section_content": "Various hematologic malignancies represent optimal targets for the exploitation of ChTCR, because of the expression of specific antigens on the surface of tumor cells, for which monoclonal antibodies are available. 24,25hus, CD19 and CD20 have been targeted for B-cell lymphoid tumors (acute lymphoblastic leukemia-ALL, lymphomas and chronic lymphocytic leukemia-CLL), CD33 for myeloid leukemia, and CD30 for lymphomas. ChTCR targeting CD19 certainly represents the best example of application of this strategy for hematologic tumors and also gives an interesting historical perspective of the evolution of this technology. 24,25The first requirement to redirect T cells towards a selected tumor target is the identification of an appropriate molecule, which is selectively expressed on cancer cells.With regard to lymphoid tumors of B-cell origin, CD19 is an ideal target, since it is present on virtually all leukemia cells in almost all cases. 16,25Among hematopoietic cells, CD19 is expressed only on cells belonging to the B-cell compartment.For this reasons, several groups have investigated its use in both in vitro and in vivo animal models. ][27][28][29] The initial construct was composed of a CD19-specific single-chain immunoglobulin extracellular targeting domain, fused to a CD3-zeta intracellular signaling domain.It was shown that CD19-redirected CTL were capable of potently killing primary B-ALL blasts and also of producing Th1 cytokines and were consistently proliferating after recognition of the targeted molecule. 26,27With the aim of translating this approach into a clinical strategy for relapsed B-ALL, Jensen's group recently focused on umbilical cord-blood transplanta-tion (UCBT), designing a method that complies with principles of current GMP for phase I clinical trials to ensure the identity, purity, potency and safety of the cellular product.UCBT-derived T cells were efficiently redirected towards the CD19 + tumor target cells (including B-ALL) by electroporation with a plasmid encoding the CD19-ChTCR, also containing a suicide gene (the herpes virus 1 thymidine kinase, HSV-1 TK, gene). 28ells were efficiently expanded in relevant numbers for clinical intervention and afterwards cryopreserved for quality controls.CD19-ChTCR-expressing UCBTderived T cells were capable of CD19-specific killing activity and cytokine secretion in vitro, as well as inducing regression of CD19 + tumors in NOD/SCID mice, and being selectively eliminated in vivo after administration of gancyclovir. 28This approach has recently been extended to autologous cell therapy for patients affected by follicular lymphoma, showing that ex vivo cell expansion from cryopreserved cell banks was sufficient to produce doses of between 5×10 9 and 1×10 10 engineered T cells for each cycle of production. 29This manufacturing strategy is therefore suitable for producing gene-manipulated T cells for phase I clinical trials in the context of B-cell lymphoid malignancies. [15][16][30][31][32] The most relevant findings obtained by this group are: i) the demonstration that the CD3-ζ signaling domain is not sufficient per se to guarantee complete and prolonged activation of manipulated T cells and that the integration of the signal transduction domain of the co-stimulatory molecule CD28 enhances the proliferative properties of the gene-modified T cells; ii) the fact that dual-specific T cells offer an improved chance of obtaining a larger in vivo expansion of engineered T cells and a long-lasting maintenance of a CD19-redirected T-cell memory pool.In particular, they used EBV-specific CTL.In fact, EBV infection is very common in humans and triggers the generation of high levels of EBV-specific CD4 + T helper and CD8 + cytotoxic T cells.Dual-specific T cells recognize EBV-infected cells through their conventional native T-cell receptor and the leukemia CD19 + target through the artificial ChTCR.Using this stratagem, the engagement of the native TCR in vivo by recurrent EBV infections is capable of constantly stimulating the ChTCR -redirected T cells, assuring appropriate activation of all pathways of Instead of manipulating conventional T cells, Campana and colleagues (St.Jude Children's Research Hospital, Memphis, Tennessee, USA) have explored the use of natural killer (NK) cells. 33,34NK may improve the therapeutic potential of allogeneic hematopoietic transplantation, but numerous published data have demonstrated that their efficacy is strongly diminished by the presence of various inhibitory HLA types.The authors therefore transduced the CD56 + CD3 -NK cells with a chimeric receptor directed against CD19.Relevant numbers of NK cells can be obtained in a relatively short time by culturing peripheral blood mononuclear cells with K562 cells expressing the NK-stimulatory molecules 4-1BB ligand and interleukin 15.Anti-CD19 ChTCR-expressing NK cells have a markedly enhanced capacity to kill B-ALL blasts.This model also corroborates the necessity to add a co-stimulatory signal to the CD3-ζ domain.Addition of 4-1BB is, in fact, followed by increased cell activation, with subsequent secretion of interleukin-2 and interferon-γ. Using the same retroviral vector kindly provided by Campana and colleagues, our group has recently investigated the efficacy of the CD19-ChTCR in the context of B-cell malignancies, transducing ex vivo expanded cytotokine induced killer (CIK) cells, which are enriched in CD3 + CD56 + CD1d-unrestricted NK-T cells. 35Such cells present peculiar characteristics that render them an attractive target for leukemia immunotherapy, since they have the intrinsic capability of reaching leukemiainfiltrated tissues, primarily the bone marrow.Our results show that CD19-ChTCR-expressing CIK cells not only become capable of efficiently killing otherwise resistant B-ALL cells, but also present high expression levels of adhesion molecules (CD49d and CD11a) and chemokine receptors (CXCR4, CCR6 and CCR7).They also show robust in vitro chemotactic activity towards their corresponding ligands, prominent adhesion and transmigration across endothelium and metalloproteases-dependent invasion of basement membrane in response to CXCL12.All this reflects their potential capability to migrate into sites of B-ALL accumulation and therefore supports the hypothesis that CD19-ChTCR-redirected CIK cells are an interesting tool for B-ALL immunotherapy. 35Moreover, from the stand point of application, CIK cells are attractive because of the reproducible and straightforward method for their generation and expansion, which only requires GMPgrade cytokines.Thus, large numbers of cells can be rapidly expanded in a closed system with minimal manipulation. Like CD19, the CD20 antigen has also been chosen to target B-cell lymphoid malignancies by ChTCR. 36,37The use of the monoclonal anti-CD20 antibody rituximab induces remissions in approximately 60% of patients with relapsed follicular lymphoma; however, most patients eventually relapse despite continued expression of CD20 on lymphoma cells.A cellular immunotherapy strategy targeting CD20 + cells by ChTRC may provide a more effective mechanism for eliminating lymphoma cells than anti-CD20 antibodies.As for the CD19-ChTCR, Jensen and colleagues electroporated peripheral blood mononuclear cells with a plasmid containing a CD20-specific scFvFc:ζ ChTCR.CD8 + CTL clones were generated and showed specific killing capacity towards CD20 + target cells, including primary tumor cells from patients affected by follicular lymphoma, small lymphocytic lymphoma, splenic marginal zone lymphoma, diffuse large B cell lymphoma, and CLL. 37Cell numbers were adequate for clinical use.In view of these findings, a phase I clinical trial for relapsed follicular lymphoma is being initiated by Jensen's group. As for B-origin malignancies, myeloid leukemias can also be targeted by a ChTCR towards the myeloid antigen CD33.Recently published in vitro data showed that human NK cells can be efficiently manipulated by electroporation, transferring a humanized chimeric immunoglobulin T-cell receptor to CD33.CD33-ChTCR-expressing NK cells specifically lysed the acute myeloid leukemia cell line KG1. 38inally, an attractive molecule for targeting Hodgkin's lymphoma tumor cells is the CD30 antigen.In fact, Hodgkin and Reed-Sternberg cells express high amounts of the cell surface antigen CD30.Hombach and colleagues used a retroviral vector containing a ChTCR gene encoding for an extracellular domain consisting of the single-chain antibody fragment HRS3-scFv with specificity for the CD30 antigen.Specific binding of the CD30-ChTCR resulted in cytolytic MHC-unrestricted reactivity against CD30 + tumor cells in vitro, thus offering an attractive model for adoptive cellular immunotherapy to be used in the context of resistant Hodgkin's disease. 39,40ven though many different molecules have been explored for human hematologic malignancies and clinical trials are ongoing or about to commence, unfortunately no data are yet available in the setting of human hematologic malignancies.However, ChTCR have been used in the setting of patients infected by immunodeficiency virus (HIV).In HIV patients, T cells have been transduced to express a ChTCR containing the extracellular domain of human CD4 which is capable of linking up the viral gp120 protein in order to recognize and eliminate HIV-infected cells.Although ChTCR-transduced T cells were functionally active in vitro and homed to the HIV-infected tissues, the anti-viral efficacy of manipulated cells was negligible. 41,42In the field of tumor immunotherapy, the use of engineered T cells expressing artificial TCR specific for tumor-associated antigens has recently given promising and exciting results in the treatment of metastatic melanoma.Even though the Table 1.A summary of the current major limitations of ChTCR technology and possible ways of overcoming them to improve ChTCR efficacy in vivo. ", "section_name": "Applications of ChTCR in the context of hematologic malignancies", "section_num": null }, { "section_content": "Short-life duration of injected ChTCR-expressing T cells Construction of new-generation ChTCR by use of fully human recombinant due to the host's immune response to foreign proteins. single-chain antibodies in the extracellular binding domain. Absence of T-helper CD4 + cells to sustain long-term cytotoxic activity.Simultaneous administration of both CD4 + and CD8 + ChTCR-modified T cells. Poor T-cell activation and proliferation after antigen-binding, Modification of the intracellular domain by introduction due to limited signaling capacity. of src family kinase lck to promote the creation of a superior signal-transducing complex. Lack of appropriate T-cell co-stimulation.Addition of co-stimulatory molecules (CD28, ICOS, CD134, or CD137). Inappropriate migration into sites of tumor infiltration. ", "section_name": "ChTCR limitation Proposed solution", "section_num": null }, { "section_content": "Insertional mutagenesis after gene transfer by viral vectors.Design of safer new generation (self-inactivating, SIN) vectors, addition of suicide genes.Use of non-integrating systems (electroporation). technology is slightly different from that used for chimeric monoclonal antibody-derived artificial receptors, the concept remains the same. 43Patients affected by metastatic melanoma were treated with autologous T cells expressing an anti-MART-1 artificial TCR.Two patients treated out of 15 showed sustained levels of circulating engineered T cells following objective regression of hepatic and pulmonary metastatic lesions. 44 ", "section_name": "Use of target cells with innate properties to migrate into sites of leukemia invasion (CIK cells).", "section_num": null }, { "section_content": "Besides showing and corroborating the strength of ChTCR technology, the available in vitro and in vivo data also highlight the presence of specific drawbacks, which must be investigated in order to find solutions that ensure better in vivo results in the human cancer setting, whilst limiting any possible side effect. Limitations of ChTCR involve both clinical and technical issues.As far as concerns clinical limitations, the currently used ChTCR lack sufficient specifity for exclusive tumor antigens.This aspect is particularly evident for the CD19 antigen, whose targeting will be accompanied by elimination of normal B cells constitutively expressing this B-lymphoid associated surface molecule.The use of anti-CD19 ChTCR-redirected T cells in human subjects will, therefore, likely determine a depression of humoral antibody-mediated immunity, which would put any treated cancer patient at further risk and necessitate continuous supplementation of immunoglobulins. 16Similar considerations are valid for the CD33 antigen, whose expression is extended to various stages of myeloid-lineage differentiation.Therefore, its targeting could potentially be followed by the killing of bone marrow neutrophil progenitors, with increased risk of bacterial and fungal infections.For these reasons, new lines of research are already exploring more tumor-specific surface antigens, with virtually absent expression on normal tissues. 16s far as concerns the technical weaknesses of current ChTCR, different factors must be taken in account, as summarized in Table 1.The first issue that could easily explain the limited survival of injected ChTCRexpressing cells is the presence of foreign molecules in the chimera (mostly mouse proteins), which induce an immune host response. 14,15New generation ChTCR will contain entirely humanized proteins, thus avoiding any recognition by the immune system. 45If this limitation seems likely to be resolved rapidly by technical improvements already available, the definition of the correct activation cascade that must be included in the ChTCR structure to have a full and long-lasting activation of manipulated T cells is more complex.It seems clear, in fact, that the activation following engagement of naïve TCR gives rise to a more complex and powerful activation of T cells, which cannot be totally provided by the existing ChTCR. 46In line with these considerations, the role of co-stimulation appears to be crucial.3][14][15][16] Such improved molecules induce a better activation of manipulated target cells, with a higher rate of proliferation, higher level of secretion of interleukin-2 and prolonged survival.The signaling characteristics of ChTCR can be further improved by linking in cis more than one co-stimulatory domain or a combination of co-stimulatory and coreceptor domains to the TCR-ζ chain.Linking the CD28 with the OX40 domain has been shown to greatly enhance the functions of ChTCR-transduced cells with markedly increased proliferation, cytokine release and effector function. 32Moreover the combination of ζchain together with co-receptor (lck) and co-stimulatory (CD28) signals in a single receptor has been demonstrated to maximize ChTCR sensitivity and potency. 47,48 different strategy to guarantee an improved and totally physiological activation of the T cells is to take advantage of the presence of an activated native TCR, transducing CTL which are already specific for a viral target (the so-called dual-specific T cells, as mentioned before). 27,49,50In this condition the activation of the natural TCR should provide a powerful stimulus, maintaining the killing capacity, the proliferative activity and the durable persistence of the ChTCR-related functions, all present in the same cell.Brenner's group recently adopted EBV-specific CTL, transducing them with a ChTCR targeting the G(D2a) antigen expressed by neuroblastoma cells. 51Such manipulated cells preserve intact activity towards the viral target and also kill the neuroblastoma cells through the artificial TCR, secreting higher levels of interleukin-2 and proliferating more consistently and durably.This strategy is currently being tested in a phase 1 trial for high-risk neuroblastoma patients.Other investigators have evaluated similar approaches using CMV-specific CTL 52 or influenza virus-specific CTL. 27ne of the most delicate and discussed topics of any gene transfer approach remains the theoretical chance of inducing oncogenic mutations after DNA integration of the vector.4][55] In both cases an inappropriate activation of LMO-2 transcription factor was detected due to proximal insertion of the missing immune gene by the retroviral vector.As thoroughly discussed by Dotti et al. in their review, 16 even though this serious adverse event triggered a sort of innate fear for any approach using any viral vector for the correction of any kind of human disease in the entire scientific community, nevertheless this was the only serious event that has occurred so far in more than 40 human trials that have used integrating vectors, and may likely be explained by the peculiarity of the targeted disease (the SCID immunodeficiency) and the type of cells which were genetically manipulated (hematopoietic stem cells). 56,57In fact, considerable amounts of stem cells were manipulated with the aim of guaranteeing the correction of the immune deficit.Moreover, the transduced T cells, expressing the inserted gene, had a proliferative advantage over the unmanipulated cells.On the other hand, in all the trials that adopted gene transfer of mature T lymphocytes, no malignant transformation has ever been observed, primarily because such mature cells have a limited proliferative capacity and have already reached a stage of terminal differentiation.Obviously, this does not mean that safety considerations regarding the use of integrating vectors are not important, especially when considering (such as for transduced ChTCR-cells) that gene transfer of co-stimulatory molecules or growth-promoting molecules may improve the survival and increase the proliferation rate of gene-manipulated T cells.This automatically implies that efficient systems of suicide genes are welcome, because they constitute a back-up protection tool, which may be used in the case of unwanted proliferation. 58 ", "section_name": "Limitations and advances in designing ChTCR", "section_num": null }, { "section_content": "The above-mentioned considerations on redirecting cytotoxic T cells towards leukemia/lymphoma-associated antigens show clearly how fascinating ChTCR technology is, but also demonstrate that complex obstacles still bar the way to an effective therapy in the context of leukemias.Only a collaborative multi-center study can provide the opportunity to study the mechanisms underlying the actual limitations of this approach (both in vitro and in vivo) and to translate this kind of therapy from bench to bed-side in the context of phase 1 trials for acute lymphoid and myeloid leukemias and lymphomas.A consortium has been recently founded thanks to funds from the European Community (STREP, Specific Targeted Research or Innovation Project, Sixth Framework Programme), which connects different centers, physicians and researchers from all over Europe.The name of the project is Childhope (www.childhope.eu).The main goal of this collaborative study is to make human T cells that express ChTCR specific for the CD19 and the CD33 antigens, for lymphoid and myeloid malignancies, respectively.These vectors will be used to transduce different types of cell targets (dual-specific EBV-CTL, CIK cells, γ-δ T cells), with the aim of comparing their in vitro and in vivo efficacy and finally choosing the more potent and persistent stimulation on the artificial receptor towards the selected tumor antigen.After an initial phase, whose only scope will be to validate the potency of the manipulated cells in vitro and in vivo in animal models, the consortium will assess the safety and potency of the ChTCR-expressing T cells specific for CD19 or CD33 in phase I studies conducted in children with relapsed or refractory B-cell lymphoid leukemias, non-Hodgkin's lymphomas, or acute myeloid leukemias, respectively.With the aim of improving the safety profile of gene transfer, the consortium will evaluate alternative methods of T-cell gene transfer, such as large-scale electroporation of T cells with mRNA (as a means to transduce T cells transiently), 59 in comparison with self-inactivating vectors (SIN), which are characterized by a different pattern of DNA integration able to give a better safety profile. 60Moreover, always with the aim of maintaining a high efficiency, but concomitantly guaranteeing a way to control any possible in vivo unwanted T-cell proliferation, a suicide gene system will be adopted and different combinations will be tested.In particular, attention will be focused on the chRec-iCasp9 suicide system, whose efficiency has been recently demonstrated. 61n conclusion, even though the clinical facts of leukemia immunotherapy are few and many difficulties stand in the way of translating any experimental approach into clinical intervention, 62 the ChTCR strategy represents an attractive targeted weapon against leukemias.Technical and safety improvements are still required, and phase 1 trials will be carried out in the near future to show the feasibility of their clinical translation and, it is to be hoped, give preliminary indications about their anti-tumor efficacy. ", "section_name": "Concluding remarks: new challenges for the near future", "section_num": null } ]	[ { "section_content": "the present work was supported by AIRC, FIRC and MIUR PRIN 2005 2005065223_002. ", "section_name": "", "section_num": "" }, { "section_content": "EB and VM analysed the literature and wrote the manuscript; ED, GMPGA and AB were responsible for critical revision of the text. The authors reported no potential conflictrs of interest. ", "section_name": "Author Contributions", "section_num": null }, { "section_content": "EB and VM analysed the literature and wrote the manuscript; ED, GMPGA and AB were responsible for critical revision of the text. ", "section_name": "Author Contributions", "section_num": null }, { "section_content": "The authors reported no potential conflictrs of interest. ", "section_name": "Conflict of Interest", "section_num": null } ]
10.1038/ncomms10087	Anaplastic large cell lymphoma arises in thymocytes and requires transient TCR expression for thymic egress	<jats:title>Abstract</jats:title><jats:p>Anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma presenting mostly in children and young adults. The natural progression of this disease is largely unknown as is the identity of its true cell of origin. Here we present a model of peripheral ALCL pathogenesis where the malignancy is initiated in early thymocytes, before T-cell receptor (TCR) β-rearrangement, which is bypassed in CD4/NPM–ALK transgenic mice following Notch1 expression. However, we find that a TCR is required for thymic egress and development of peripheral murine tumours, yet this TCR must be downregulated for T-cell lymphomagenesis. In keeping with this, clonal TCR rearrangements in human ALCL are predominantly in-frame, but often aberrant, with clonal TCRα but no comparable clonal TCRβ rearrangement, yielding events that would not normally be permissive for survival during thymic development. Children affected by ALCL may thus harbour thymic lymphoma‐initiating cells capable of seeding relapse after chemotherapy.</jats:p>	[ { "section_content": "ucleophosmin-anaplastic lymphoma kinase (NPM-ALK) positive anaplastic large cell lymphoma (ALCL) is considered as a peripheral T-cell lymphoma and is associated with the t(2;5)(p23;q35) translocation, identified to be the fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM1 (ref.1).ALCL is defined by expression of the CD30 marker of activated B and T cells and, in some cases, T cell markers such as CD4 or CD8, as well as production of cytotoxic molecules such as perforin, which together with the presence of clonal T-cell receptor (TCR) rearrangements, suggests an activated T-cell origin [2][3][4][5][6] .Other studies have proposed a Treg cellular origin (owing to the generation of FoxP3 transcripts and production of IL10 and TGFb by ALCL cell lines) or more recently a Th17 phenotype (owing to production of IL17 by tumour cells) [5][6][7][8] .Whether these cellular attributes are a remnant of the cell of origin or are NPM-ALK induced remains to be fully elucidated.Despite the presumed T-cell affiliation, ALK þ ALCL rarely express TCRb or CD3e by immunohistochemistry 9 , and the CD3 signalling cascade is downregulated in an NPM-ALK-dependent manner by transcriptional and epigenetic modification 10 .More recently, we have shown that a subpopulation of ALCL cancer stem cells (CSC) express genes enriched within the gene set of early thymic progenitors (ETPs), hinting towards a primitive cell of origin 11 .Our data suggest that the seeds of ALCL are sown in the thymus, or even earlier in hemopoietic development, perhaps indicating that events in the thymus, or once the primed cells exit the thymus, might shape the final cell phenotype.To investigate this hypothesis, we carried out a thorough analysis of TCR rearrangements in human ALCL to look for evidence of NPM-ALK subverted thymic T-cell development.During normal T-cell development, TCRa VJ rearrangement is preceded by TCRb VDJ rearrangement and massive cellular expansion of thymocytes during pre-TCR b-selection.The process of b-selection is driven by the pre-TCR composed of a pre-TCRa expressed at the cell surface together with the TCRb chain.TCR immunogenetic profiles by multiplex PCR can therefore be used to identify lineage affiliation and stage of maturation arrest. Our data show that most ALK þ ALCL demonstrate immunogenetic evidence of in-frame TCR rearrangement, 25% displaying atypical TCR profiles not normally selected for during (or permissive of) TCR-ab lineage thymic development.In normal T-cell ontogeny, these events would result in a failure of b-selection and thymic cell death, suggesting that event(s) in the thymi of these patients had subverted b-selection.We therefore used the CD4/NPM-ALK (CD4NA) thymic T cell lymphoma model 12 to investigate the potential contribution of recombinase activating gene (Rag)-mediated TCR rearrangement and TCR signalling to the natural progression of NPM-ALK-driven T-cell lymphoma.In the absence of Rag-mediated TCR rearrangements, NPM-ALK can induce 'T cell maturation' to the CD4 þ CD8 þ double-positive (DP) and CD4 single-positive (SP) stages of T-cell development, allowing the formation of thymic lymphomas.We hypothesized that expression of some form of TCR might be required to initiate thymic egress and therefore backcrossed the CD4NA transgenic line to the class I-restricted Ova-specific TCR transgenic line OT1, on both a Rag-competent and Rag-deficient background 13,14 .The presence of the OTI TCR moves the site of lymphoma presentation to the periphery although tumour cells do not express a TCR, and TCR stimulation prevents lymphoid oncogenesis; as such this model more resembles human ALCL, which also rarely express a TCR 9,10 .These data suggest that peripheral ALK þ ALCL depends on transient expression of a functional TCR to enable thymic emigration of primed T lymphocytes and its subsequent downregulation once cells are in the periphery to permit transformation. ", "section_name": "N", "section_num": null }, { "section_content": "Abnormal TCR rearrangements in human ALCL tumours.As NPM expression is ubiquitous, the NPM-ALK fusion could lead to expression of ALK at all stages of thymic development 15 .To investigate whether ALCL cells have passed through the thymus while expressing NPM-ALK, we analysed the status of TCR rearrangements in human tumours.TCRd, TCRg and TCRb genes were amplified from DNA of ALCL tumours, and clonal populations identified by genescan analysis of PCR products, with an approximate sensitivity of 1-5% (Fig. 1, Table 1, Supplementary Table 1 and summarized Supplementary Fig. 1).To refine the TCR status, 180K Agilent CGH array analysis and PCR detection of clonal TCRa transcripts from complementary DNA (cDNA) using Va and Ca primers, was performed for selected cases (Table 1). A total of 57 ALK þ ALCL with at least 40% tumour cell content were analysed.T lymphoid immunohistology and cytotoxic status is summarized in Table 1 and detailed in Supplementary Table 1.Overall, clonal TCRg VJ rearrangements were identified by PCR in 49 (75%), TCRb VDJ in 33 (58%) and TCRd VDJ in 11 (19%) cases (Table 1).On the basis of these data, we classified the ALCL into four distinct categories based on the TCR rearrangement profile observed: TCR germline (GL), TCRab, TCRgd and TCRg only. Eight (14%) tumours demonstrated no significant evidence of clonal TCR rearrangement by PCR or TCRa/d CGH, despite at least 50% tumour cell content (Fig. 1b,d, Table 1 and Supplementary Table 1) and were categorized as TCR GL.As such, they represent the molecular equivalent of null ALCL (although six out of eight cases expressed cell surface markers associated with a T-cell phenotype (Supplementary Table 1)). Tumours were classified as TCRab if a clonal TCRb VDJ rearrangement was detected by PCR on at least one allele, in the absence of TCRd VDJ rearrangement, consistent with bilateral TCRd deletion during TCRa rearrangement.This was the predominant category (32 out of 57; 56%; Fig. 1d).The vast majority (16 out of 17 tested) of TCR-ab ALK þ ALCL demonstrated clear evidence of TCRa VJ on at least one allele by CGH and clonal TCRa transcripts were identified by reverse-transcriptase PCR (RT-PCR) in 10 out of 15 CGH-positive cases (Fig. 1b).Unexpectedly, TCRb rearrangements, despite being clearly detected by PCR, were only detected by CGH in a minority of cases (3 out of 17, 18%), with two additional cases (ALCL19 and 07) demonstrating heterogeneous deletions (Fig. 1c).This was not owing to insufficient resolution of the Agilent 180K arrays, as even TCRb rearrangements involving 5 0 Vb were not seen and repeat CGH using Affymetrics high density Cytoscan arrays (2.7 M probes) in three TCRb PCR VDJ þ /CGH À TCRab cases confirmed clear evidence of TCRa VJ rearrangement, but no detectable TCRb VDJ, with only one case demonstrating an expected clonal incomplete TCRb DJ (ALCL24) (Supplementary Fig. 2). Sequence analysis of clonal PCR products demonstrated at least one in-frame TCRa rearrangement in eight out of nine TCRab cases, with three cases demonstrating two in-frame rearrangements.Sequencing of the TCRb VDJ rearrangements from 16 TCR-ab ALCL (Supplementary Table 1) identified a mono-allelic, in-frame rearrangement in all but one case, ALCL 26, notably one of the rare cases to be positive by TCRb CGH.Despite this evidence of a functional TCRab, none of the 16 ALCL analysed by immunohistochemistry demonstrated detectable TCRb expression on tumour cells (Supplementary Fig. 3A).Thirteen TCRg alleles were sequenced from eight TCRab cases, but as expected, only two (15%) alleles were in-frame (Supplementary Table 1).Taken together, TCRab tumours demonstrate clear evidence of in-frame clonal TCRa rearrangement but only minor, in-frame TCRb rearrangements which are detected by PCR but rarely by CGH array, in keeping with heterogeneous, on-going TCRb rearrangements within the TCRa clone, or with reactive, tumour infiltrating lymphocytes.To determine whether these on-going rearrangements bore any clonal ancestry, we cloned and sequenced the TCRb rearrangements from ALCL19, but none of the minor rearrangements shared any apparent ancestral TCRb CDR3 DJ sequence. Six cases demonstrated clonal TCRg rearrangements but no TCRd rearrangement and, at the most, incomplete TCRb rearrangement (three monoallelic DJ), and were labelled TCRg-only (Fig. 1b,d).Surprisingly, the TCRa locus was found to be rearranged in two out of four cases by CGH and clonal TCRa VJC transcripts were detected in five out of six by RT-PCR (Fig. 1b, Supplementary Fig. 2,ALCL30 and Table 1).Strikingly, all six TCRa rearrangements sequenced from these five ALCL were in-frame, despite the apparent absence of clonal TCRb VDJ rearrangement.This was not due to false negative TCRb PCR results, as 180K CGH analysis did not identify TCRb rearrangement in four cases tested (Supplementary Table 1) and high resolution 2.7M Cytoscan analysis of ALCL30 only identified the expected TCRb DJ (Supplementary Fig. 2B).Only one (ALCL23) had no evidence of TCRa rearrangement by RT-PCR and CGH.As such, it resembled a TCR GL ALCL with an isolated 'immature' out-of-frame VgfI-JP1/2 TCRg rearrangement.In the absence of TCRb, TCRa could, at least hypothetically, pair with TCRg, as all cases had undergone at least mono-allelic rearrangement, but only one of the six TCRg alleles sequenced was in-frame.As such, five out of six TCRg-only cases demonstrate potentially functional TCRa rearrangement in the absence of TCRb rearrangement and only differ from TCRab cases by the absence of a TCRb clonal VDJ peak by PCR. Eleven cases (19%) were classified as TCR-gd since clonal TCRg VJ and TCRd VDJ rearrangements were detected (Fig. 1b,d).This category demonstrated a uniform genotype, with 10 out of 11 demonstrating a Vd1-Jd1 /Dd2-Jd1 genotype and an in-frame TCRg and TCRd rearrangement on at least one allele (Supplementary Table 1).The 180K CGH profiles confirmed only TCRd but no TCRa or TCRb rearrangements in the six cases tested (Supplementary Table 1 andTable 1).They all demonstrated an incomplete TCRb DJ on at least one allele (below the resolution of CGH detection).ALCL36 also demonstrated a complete, in-frame TCRb VDJ rearrangement but was classified as a TCR-gd since the TCRd rearrangements were bilateral, the clonal TCRg was in-frame and no TCRa transcripts were detected by RT-PCR.Thus, as for TCR-ab ALCL, TCR-gd ALCL have immunogenetic evidence of a potentially functional TCR. In summary, PCR and CGH analysis of TCR rearrangements in ALK þ ALCL showed that two-thirds (TCR-ab and TCRgonly) demonstrate a major in-frame TCRa clonal rearrangement, which is not accompanied by a comparable major TCRb clonal population but by minor TCRb clonal populations that are predominantly in-frame and may originate within or independently to the TCRa clonal population and are not associated with detectable TCRb protein by immunohistochemistry.The remaining third are divided between immunogenetic 'Null-ALCL' (TCR germline, 14%) and immunogenetic TCR-gd ALCL (19%). We therefore hypothesized that, in keeping with our prior suggestion that NPM-ALK may replace the TCRb signalling cascade 16 , NPM-ALK may allow an immature hematopoietic cell to bypass thymic b-selection to a stage when TCRa VJ rearrangement becomes possible, thus enabling abnormal T lymphoid differentiation and implying a thymic origin to at least a proportion of ALCL.To evaluate whether ongoing recombinase activity could occur in established ALK þ ALCL tumours, we looked for RAG1 transcripts in 52 cases, but all were negative, as were all the three ALCL cell lines tested in contrast to the immature T-ALL cell lines arrested at a thymic stage of development (Supplementary Fig. 3B). ", "section_name": "Results", "section_num": null }, { "section_content": "To elucidate whether NPM-ALK expressing thymocytes might develop and eventually undergo transformation in the absence of a functional TCR rearrangement, we generated transgenic mice expressing NPM-ALK at all stages of thymic development which lacked the rag2 gene (Fig. 2a).RAG2 is required for TCR rearrangement hence rag2 À / À mice have small thymi devoid of mature T cells but contain normal numbers of T-cell progenitors blocked in development at the DN3 stage.We analysed the thymi of both CD4NA/RAG þ / þ and CD4NA/RAG2 À / À mice before the presentation of overt tumours.In CD4NA/RAG þ / þ mice at 5 weeks of age, there is an accumulation of phenotypic DN3 cells.(Fig. 2b,c, absolute cell counts are shown in Supplementary Table 2).There was also an increase in the percentage of the total thymic population expressing the cell surface marker CD117, a protein whose expression is switched off at the DN3/4 stage of thymic development (Fig. 2b,c).These data suggest a delay in T-cell development at this stage induced by NPM-ALK activity and consistent with the time at which b-selection occurs. In the CD4NA/RAG2 À / À mice, the cellularity of the thymus was increased significantly over numbers seen in RAG2 À / À mice but not to the levels observed in wild-type mice (Fig. 2e, absolute cell counts shown in Supplementary Table 3).Cell numbers observed in the RAG2 À / À line are consistent with previous reports 13 .In CD4NA/RAG2 À / À mice, the numbers of DN3 cells were over-represented compared with wild type mice.Notably, the majority of thymocytes in CD4NA/RAG2 À / À were DP (Fig. 2e,f).These data indicate NPM-ALK can promote the bypass of the b-selection checkpoint.In further support of these data, 32% less DN4 cells isolated from CD4NA mice express TCRb on the cell surface compared with wild-type littermate control mice (Supplementary Fig. 4A).Furthermore, DN3 cells isolated from the CD4NA/RAG2 À / À transgenic mice expressed cell surface Notch1, consistent with apparent maturation of thymocytes beyond the DN3 stage (Fig. 2g) as well as upregulation of the nutrient transporters CD98 and CD71 required to meet the metabolic demands of developing thymocytes (Supplementary Fig. 4B).Indeed, CD98 and CD71 upregulation are usually only seen in the presence of signalling by Notch1 and the pre-TCR, suggesting that NPM-ALK not only induces Notch1 expression but also compensates for absence of the pre-TCR 17 . The CD44 hi population is the precursor of tumour growth.An abnormal population of thymocytes is detectable in CD4NA transgenic mice at later ages, when thymic cellularity is still within the normal range.CD4/CD8 mature T cells express the immature cell surface marker CD117 as well as CD44, the latter being normally restricted to DN1/2 thymocytes (Supplementary Fig. 5A).Whether the increase in CD44 hi cells is a remnant of expression on DN1/2 cells or NPM-ALK induced upregulation of CD44 is unclear, though the latter explanation is supported by experiments in which NPM-ALK is capable of driving expression of luciferase from the CD44 promoter (Supplementary Fig. 5B). These data suggest that NPM-ALK drives asynchronous TCRb independent proliferation of DN3-like cells and the maturation to DP cells, without loss of DN1/2 characteristics such as expression of CD44 and CD117.This may provide an oncogenic advantage to this population, which is not subjected to thymic TCR-mediated selection.Indeed, this CD44 þ population gave rise to thymic tumours when transplanted intravenously into recipient mice, again consistent with an origin in ETPs in this model system (Supplementary Fig. 5C). NPM-ALK drives TCR-independent thymic tumour formation.Despite the absence of TCR rearrangement, the CD4NA/ RAG2 À / À mice produce thymic tumours consisting of cells reminiscent of the double-positive stage of T-cell development (Fig. 3a, Table 2).However, tumours arising in the CD4NA or CD4NA/RAG2 À / À lines display the same histopathology, consisting of NPM-ALK-expressing diffuse, homogenous sheets of medium-sized cells with complete effacement of the normal thymic architecture (Fig. 3b).As expected, the CD4NA/ RAG2 À / À tumours, despite expressing CD4 ± CD8 do not display TCR rearrangements (Fig. 3c).These data demonstrate that TCR rearrangement is not necessary for cellular transformation in the thymus, although the development of tumours in CDNA/RAG2 À / À mice compared with CD4NA is delayed, suggesting that this process may enhance tumorigenesis (Fig. 3d).NA in the absence of TCR rearrangement therefore allows asynchronous thymic development to the CD4/8 DP stage and development of cortical thymic lymphoma. Murine tumours are always restricted to the thymus, whereas ALCL in humans present for the most part in the periphery and are often extranodal (although mediastinal involvement has been observed in as many as 50% of human cases 18 ).It is therefore clear that if the tumours initiate in the thymus, something must contribute to thymic egress.During normal thymic maturation, this is normally provided by surface expression of a TCR/CD3 complex.ALCL is, in contrast, characterized in both humans 9 and our murine CD4NA/RAG2 À / À model by complete absence of a TCR/CD3 complex, thus potentially explaining the failure to exit the thymus, at least in mice.This is not always the case, as CD4NA mice can, in rare cases, present with peripheral tumours (in our hands one out of 50 CD4NAmice develop tumours in the peripheral lymph nodes).In addition, our data show that the majority of human ALCL demonstrate immunogenetic evidence of an in-frame TCRab or TCRgd, suggesting prior expression of a functional TCR.We therefore restored TCR expression in the mouse model by introducing a TCR transgene. Presence of an OT1 TCR results in peripheral T-cell lymphoma.The OT1 TCR (Va2) recognizes ovalbumin (ova) residues 257-264 (SIINFEKL) in the context of H2k b (ref.14).In the absence of ova, the CD4NA/RAG2 À / À /OTI mouse line do not develop lymphoid tumours, but instead present after a longer latency with (OT1 TCR negative) gastrointestinal stromal tumours, hepatocellular carcinomas and sarcomas, even though OTI TCR-positive T cells exist in the periphery (Supplementary Fig. 6, Supplementary Table 4).In contrast, the RAG competent CD4NA/OT1 line do produce ALK þ lymphomas, with a peripheral rather than thymic presentation, and a mean survival of 181 days (versus 88 days for the CD4NA mice (P ¼ 0.0001, Student's t-test); Fig. 4a).Whether this delay in tumour development is a true difference or purely a result of belated tumour detection (the thymic CD4NA tumours induce laboured breathing, whereas peripheral tumours in CD4NA/OTI mice can be more difficult to detect owing to asymptomatic growth) is not clear.However, analysis of the DN3 population in CD4NA/OTI mice in comparison with CD4NA mice showed increased cellular proliferation in the former, which could potentially reduce time available for the acquisition of tumour-inducing secondary genetic events (possibly RAG-mediated) allowing thymic escape and transformation in the periphery (Supplementary Fig. 7).However, the CD4NA/OTI mice have apparently normal T-cell development profiles although total thymic cellularity is increased (Supplementary Fig. 8A-C).Notably, the peripheral tumours in the CD4NA/OTI line more closely replicate the histopathological presentation of the human disease (Table 2, Fig. 4b).All the tumours are positive for ALK staining by immunohistochemistry and histological analysis showed cells with eosinophilic cytoplasm and reniform nuclei, resembling the classic 'hallmark' cells of human ALCL (Fig. 4b) 19 .Despite the presence of RAG in these mice, the tumours did not display endogenous TCRb rearrangements, suggesting that they arose from the thymic populations expressing the OTI transgenic TCR, which is known to suppress endogenous rearrangements mediated by RAG (Fig. 4c).Expression of the OTI TCR (still in the absence of ova) would signal thymic emigration in the absence of other TCR rearrangements.However, the tumours rarely expressed the transgenic Va2 TCR on the cell surface (only one out of 10 mice analysed; Fig. 4d, Table 2) even though Va2 expressing T cells coexist in unaffected organs of tumour-bearing mice (Fig. 4e) and are present in the periphery of pre-tumorigenic mice (Supplementary Fig. 6B), suggesting that its expression had been lost.Intracellular CD3 could be detected, but surface CD3 was not expressed on the tumours and there was an absence of intracellular Va2 TCR even though OTI transcripts could be detected (Supplementary Fig. 8D,E).This 'null' cell surface phenotype (Fig. 4d) is strikingly reminiscent of human ALK þ ALCL.It has been reported that the IL2R and molecules downstream of TCR-ligation induced signalling are silenced by epigenetic mechanisms in ALCL 10,20 , suggesting that TCR-induced signalling acts as a tumour-suppressive mechanism. As the CD4NA/RAG2 À / À /OTI transgenic mice do not develop lymphoid tumours, it was possible that RAG was acting as an essential tumour promoter in the NPM-ALK/OTI mouse.RAG1 transcripts are not produced by human ALCL tumours (Supplementary Fig. 3B) (even though RAG2 transcripts are detectable in our murine NA/OTI tumours, Supplementary Fig. 8E).RAG is normally lost with expression and cognate signalling of a functional TCR, so its absence in human ALCL is compatible with mature post-thymic cells but does not exclude RAG expression at an earlier stage of ALCL development.Alternatively, B-cells (which are not produced on the RAG À / À mouse background) might be an essential contributor to tumour development although there was no significant B-cell infiltration in tumours arising in the CD4NA/OTI mice (Supplementary Fig. 8F). To assess the role of TCR-Ag interaction in peripheral ALCL development, we exposed NA/OT1 RAG competent mice to MHV-OVA (MHV68; ref. 21).Strikingly, this abrogated ALCL development in favour of hepatocellular carcinomas and sarcoma, as seen in the NA/RAG2 À / À /OT1 mice (Fig. 4f, Supplementary Table 4).This demonstrates that cognate peripheral TCR signalling in NPM-ALK expressing cells is not compatible with ALCL development and/or survival.As expected, the OTI Va2 TCR was not expressed on the surface of tumours developing in these mice, whereas it was present in unaffected haematological tissue (Fig. 4g). Taken together, these data demonstrate that NPM-ALK permits bypass of thymic b-selection, but that TCR signalling is required for thymic egress and development of peripheral ALCL, the latter of which is dependent on subsequent abrogation of the TCR (Supplementary Fig. 9).Indeed, the analysis of 16 human ALK þ ALCL by double staining for CD30 and the TCRb chain showed an absence of tumour cells co-expressing both proteins, again suggesting that the presence of a TCR is not compatible with tumour cell survival in the presence of NPM-ALK (Supplementary Fig. 3A). ", "section_name": "NPM-ALK can induce thymic 'T cell maturation'.", "section_num": null }, { "section_content": "It has been notoriously difficult to recreate the clinical and immunophenotypic features of human ALCL in transgenic mouse models 12,[22][23][24][25] .We now demonstrate that CD4-driven NPM-ALK can lead to both bypass of the RAG2 À / À -mediated murine DN3 thymic maturation block and to the development of cortical thymic lymphoma.However, TCR expression is required for thymic egress and development of peripheral tumours which, for the first time, histologically resemble human ALK þ ALCL.The fact that these peripheral murine tumours cannot develop in the presence of ova-induced stimulation of the transgenic TCR and have lost endogenous TCR expression is strikingly similar to the widely recognized absence of TCR expression and signalling 9,10 , despite prior clonal TCR rearrangement, in human ALCL, and suggest that persistent co-existence of TCR and NPM-ALK-driven signalling in the periphery is not compatible with ALCL development and/or survival.Immunogenetic analysis of human ALCL demonstrated that the majority have major in-frame, potentially selected, TCRa receptor rearrangements in the absence of comparable TCRb rearrangements, in keeping with the absence of detectable TCRb protein, compatible with replacement of TCRb by NPM-ALK in the development of classical ALCL.NPM-ALK allowed cells to undergo asynchronous differentiation through thymic b selection in the absence of TCRb rearrangement in the RAG2 À / À model, with expression of Notch1 (unlike rag2 À / À mice) and its downstream CD98 and CD71 nutrient transporters 26 but maintenance of CD44 and CD117 expression, in keeping with previous identification of an ETP transcriptional signature in ALCL 11 .This indicates that the initial stages of ALCL development occur in the thymus and is compatible with the identification of a mediastinal mass in 50% of pediatric ALCL and high-level expression of Notch1 in ALCL 18,27 .As such, NPM-ALK signalling must be able to at least partially mimic pre-TCR signalling and allow maturation to TCRa rearrangement, including in the apparent absence of major clonal TCRb rearrangement, as identified in 67% of (TCRab and TCRg-only) human ALCL.NPM-ALK does not appear to prevent on-going TCRb rearrangement in human ALCL, as many cases demonstrate clonal populations by PCR (but not by high-resolution CGH) and as such are probably present in 1-20% of the DNA.It is not possible to determine whether these minor rearrangements originate within the ALCL population or represent tumour infiltrating reactive T lymphocytes, but we favour the former as tumour infiltrating reactive T lymphocytes should be detectable with TCRab-specific antibodies by immunohistochemistry, which was not the case. The delayed onset of lymphoma development in the NA/RAG2 À / À model and its absence in the NA/RAG2 À / À / OT1 model would suggest that TCR signalling (or other RAG induced recombination) contributes to lymphomagenesis, although whether this is due to other, off-target RAG effects remains to be investigated.RAG activity in T and B lymphoid precursors carries the risk of creating recombinase-mediated abnormalities that might contribute to lymphomagenesis, as recognized in acute lymphoblastic leukemia 28 .An alternative explanation might lie in the ability of RAG proteins to induce DNA strand breakage at (cryptic) recombination signal sequences, which can lead to the production of oncogenic chromosomal translocations.It is unlikely that the absence of B lymphocytes is the explanation for failure to develop lymphoma in the absence of RAG, as there was no significant B-cell infiltration in tumours arising in CD4NA/OTI mice. In the present model, RAG is dispensable for thymic tumours arrested before positive and negative selection in DP cortical thymocytes, but is required for peripheral tumours, as evidenced by the absence of lymphoma in NA/RAG À / À /OT1 mice, in keeping with a role for recombinase-mediated events in ALCL development.The fact that development of peripheral ALCL in the recombinase competent NA/OT1 mice was associated with loss of TCR transgene expression, and that induction of constitutive TCR signalling by exposure of NA/OT1 mice to ova abrogated peripheral ALCL development, suggest that TCR-induced signalling acts as a tumour-suppressive mechanism in the presence of NPM-ALK in peripheral T cells.'Immunesurveillance' or tumour suppressor function in ALCL has been ascribed to IL2Rg signalling and NPM-ALK downregulates TCR-related signalling molecules by kinase-dependent, STAT3mediated, gene transcription and/or epigenetic silencing 10,20 . One possible explanation is that TCR þ ALK þ cells can leave the thymus and survive in the periphery, but that TCR signalling triggered by antigen stimulation leads to clonal deletion in the presence of oncogenic ALK.Abrogation of TCR signalling, as seen in human ALCL, and re-created in the RAG-competent NA/OT1 'silent' transgene in the absence of ova, would leave ALK signalling unopposed, thus favouring lymphoproliferation.It is possible that Ag (or other) T-lymphoid stimulation within a certain context, in addition to inducing expression of ALCL-hallmark activation markers such as CD30, perforin and Granzyme B (ref. 3), and initial TCR signalling, may in some way subsequently trigger TCR silencing and unopposed ALK signalling.In keeping with this, an antigenic stimulus triggering a strong immune response before ALCL onset, such as systemic ALK-positive ALCL skin lesions occurring after an insect bite, has been described 29 .NPM-ALK can also lead to ALCL development in T-lymphoid subsets other than TCRab precursors, as 19% of human cases expressed a uniform TCRgd immunogenetic profile, with thymic type Vd1-Jd1 rearrangements 30 .Naive TCRgd cells produce IL-17, and a TH17 profile has been identified by transcriptional profiling in ALCL 31 .Eight ALCL (14%) expressed combinations of CD2, CD4, CD5 or CD7 but no detectable TCR rearrangements and as such correspond to the immunogenetic equivalent of null ALCL, which could result from transformation of NK or other innate immune subsets.These cells respond to an inflammatory cytokine environment rather than being classically TCR/MHC-restricted.However, the majority (80%) of human ALCL do demonstrate immunogenetic evidence of at least transient TCR expression and selection during ALCL development. In normal TCRab lineage thymocytes, absent or excessive TCR signalling leads to cell death, as part of positive and negative selection of the TCRab repertoire.Our data suggest that ALK-driven signalling in the absence of RAG can replace an absent thymic pre-TCR proliferative signal, with uncontrolled cortical thymocyte expansion (as seen in the NA/RAG À / À model) as there is no mechanism for TCR-mediated negative selection.Co-existence with a functional TCR allows thymic egress but subsequent cell death in the presence of simultaneous TCR and NPM-ALK signalling, by a process analogous to thymic negative selection.As such, the TCR acts as a tumour suppressor, which must be downregulated for development of classical, peripheral ALCL.These observations clearly need further experimental investigation of the precise mechanisms underlying peripheral counter-selection but the current data describe a murine model, which for the first time closely resemble human ALCL and which will be useful for evaluation of novel therapeutic agents. Furthermore, this work raises the possibility that children affected by ALCL harbour 'lymphoma-initiating cells' in the thymocyte population that could contribute to relapse after chemotherapy has apparently cleared the disease.Previously, we have shown that ALCL-propagating cells express a gene signature associated with an ETP in keeping with a presumed thymic origin as described in the current model 32 .Indeed, NPM-ALK transcripts have been detected in 2% of newborn cord blood 33 and expression of NPM-ALK is regulated by the endogenous NPM1 promoter, which drives ubiquitous expression of NPM1 including in thymic progenitors (Supplementary Fig. 10).Thus, it is not unreasonable to speculate that NPM-ALK is likewise expressed in this cellular population. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Human ALCL biopsies.Tumour biopsies were obtained with informed consent at diagnosis from 57 ALK-positive ALCL centralized in the Department of Pathology Universitaire du Cancer in Toulouse in accordance with institutional review board-approved protocols (DC 2009-989) and in accordance with the Helsinki Declaration of 1975, as revised in 2000.All but two (one soft tissue mass, one colon tumour) were lymph node biopsies.Among the 57 patients, 35 were male and 22 female.The study group included 48 paediatric ALCL (less than 18 years old) and 14 adult ALCL.The diagnosis of ALCL was made on formalin-fixed paraffin-embedded tissues and based on morphologic and immunophenotypic criteria, as described in the last WHO classification 34 , using a large panel of monoclonal antibodies against CD30/BerH2, ALK, EMA, several T cell (CD2, CD3e, CD4, CD5, CD7, CD8, CD43) and B-cell markers (CD20, CD79a) and cytotoxic molecules (Perforin, Granzyme B, TiA1).Cases were classified as T/NK lineage if they reacted with one or more antibodies against the T-or NK-cell antigens CD2, CD3e, CD4, CD5, CD7, CD8, CD43 or cytotoxic molecules and lacked reactivity for the CD20 and CD79a B-cell-associated antigens.A null phenotype was assigned to cases that did not express either T/NK-or B-cell-associated markers.All the cases were reviewed by an international pathology panel.Fifty cases expressed NPM-ALK protein, four TPM3-ALK and two ATIC-ALK; in one case, the ALK partner was not identified. Frozen tumour samples with informed consent were retrieved from CHU de Toulouse tumour tissue bank, in accordance with institutional review board-approved protocols (DC 2009-989) and in accordance with the Helsinki Declaration of 1975, as revised in 2000.The percentage of malignant cells was assessed on frozen sections by ALK1 or CD30 staining and was greater than 40% for all the selected cases.Total DNA extraction was performed from 10 frozen sections of tumour biopsies (10 mm thickness each), using the QIAamp DNA Mini kit (Qiagen, Courtaboeuf, France), following the manufacturer's protocol.Total RNA extraction was performed from 40 frozen sections of tumour biopsies (5 mm thickness each), using Trizol total RNA isolation reagent (Invitrogen, San Diego, USA) following the manufacturer's protocol.The concentration was quantified by NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, USA).RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent, Massy, France).DNA integrity was assessed by 6% polyacrylamide gel electrophoresis.Quantification of RAG1 transcripts was performed as previously described 35 . Human thymi were obtained as surgical tissue discards from children, with informed consent from the parents and the ethical review board of Necker Enfants Malades Hospital at Paris Descartes. Genescan analysis of human TCR rearrangements.The TCRg multiplex PCR were performed as described 36 .Briefly, 500 ng DNA was amplified for 35 cycles with 0.25 mM each primer, 2.5 mM MgCl 2 and 1 U Taq Gold (Life Technology). The TCRb and TCRd multiplex PCRs were developed within the Biomed-2 BMH4-CT98-3936 Concerted Action @JJM 37 .Briefly, 100 ng DNA was amplified for 35 cycles in the presence of 0.2 mM each primer, 2 mM MgCl 2 and 1 U Taq Gold (Perkin Elmer).TCRb gene configuration was assessed with a three-tube multiplex PCR, two of which contained 27 Vb family-specific upstream primers with either nine (PCR A) or four (PCR B) downstream Jb primers (Supplementary Table 5).The third (PCR C) contained all 13 Jb primers and Db1 and Db2 upstream primers (Supplementary Table 5).TCRb and TCRd PCR products were analysed by heteroduplex analysis and Genscan analysis of multiplex fluorescent PCR by separation of fluorochrome-labelled single strand (denatured) PCR products in a capillary sequencing polymer and detection via automated laser scanning.This results in a Gaussian distribution of multiple peaks representing many different PCR products in the presence of polyclonal rearrangements, but a discrete, narrow peak if a clonal population represents at least 1-5% of the DNA. The large number of Va and Ja segments precludes PCR detection of clonal rearrangements from DNA, although TCRa VJC transcripts can be detected from cDNA.TCRa rearrangements were amplified from cDNA using Ca and Va primers in five multiplex RT-PCR reactions 38 .RNA was isolated using an RNeasy kit according to the manufacturer's instructions (Qiagen, Courtaboeuf, France) and 1 mg of total RNA was converted into cDNA using SuperScript III RT (Invitrogen, San Diego, USA).RNA quality and quantity was assessed relative to the ABL housekeeping gene, according to protocols developed within the Europe against Cancer network 39 on an AB 7900HT (Applied Biosystems, Foster City, CA, USA).RNA (20 ng) was amplified in each PCR with 1 U of Hotstart Taq (Qiagen, Courtaboeuf, France), 10 mM dNTPs, 2 mM of MgCl 2 , 20% Q solution, and 10 pmol of each primer.The Taq polymerase was activated for 15 min at 95 °C, followed by 37 cycles of 94 °C for 30 s, 63 °C for 45 s and 72 °C for 1.5 min.The final elongation step was 72 °C for 10 min.The negative control consisted of RNA isolated from peripheral blood mononuclear cells from donors and the positive control was RNA isolated from the Jurkat cell line.Primers sequences used for the TCRA transcript amplification are listed in Table 3. CGH analysis of TCR rearrangements.DNA at diagnosis were analysed by array-comparative genomic hybridization (array-CGH, Agilent human CGH MicroArray, 180K) and by High Density Affymetrics Cytoscan (2.7 million probes) according to the manufacturer's recommendations.Deletion of TCR loci, as a reflection of V(D)J recombination, were defined by the CGH log 2 copy number ratio, with deletion/rearrangement on one allele defined as 0.5 to 1.5, whereas log 2 copy number ratios o1.5 were defined as biallelic deletions/rearrangements.Data are accessible on the NCBI GEO database under GSE67131. Transgenic mice.The CD4/NPM-ALK (CD4NA) mice were generated as previously described (kindly provided by Professor G. Inghirami, University of Turin, Italy) 12 .NPM-ALK/OTI (CD4NA/OTI) mice were generated by crossing the CD4NA line to the OT1 transgenic T cell line (kindly provided by Professor G. Griffiths, University of Cambridge, UK) 14 .NPM-ALK/RAG2 À / À (NARAG2 À / À ) mice were generated by crossing the CD4NA line to the RAG2 À / À line (kindly provided by Professor C. Rudd, University of Cambridge, UK).NPM-ALK/RAG2 À / À /OT1 (NA/OTI/RAG2 À / À ) mice were generated by crossing the NARAG2 À / À line with the OT1 transgenic T cell line that had been backcrossed to the RAG2 À / À line.OTINA mice were exposed to 2 Â 10 4 p.f.u.MHV-Ova (MHV68), intranasally on three occasions at monthly intervals starting at 6 weeks of age.Virus was prepared as described in ref.21 and was provided by Dr P. G. Stevenson, University of Cambridge.All the mice were housed under specified pathogen free conditions in ventilator cages within a barrier facility at the University of Cambridge and were maintained on a C57/Bl6 genetic background and were analysed at 4-12 weeks of age unless indicated.All the procedures were conducted under UK Home Office license 80/2630 at the University of Cambridge according to UKCCR guidelines. Genotyping of the mice was performed by PCR on DNA extracted from ear biopsies, and the PCR products were separated by agarose gel electrophoresis as described previously 12,40,41 using the following primers: OT1 FWD 5 0 -ACGTG TATTCCCATCTCTGG-3 0 , OTI R 5 0 -CTGTTCATAATTGGCCCGA-3 0 ; NPM-ALK FWD 5 0 -TCCCTTGGGGGCTTTGAAATA-3 0 , NPM-ALK RVS 5 0 -CGAGGTGCGGAGCTTGCTCAG-3 0 ; RAG A 5 0 -GGGAGGACACTCACTTG CCAGTA-3 0 , RAG B 5 0 -AGTCAGGAGTCTCCATCTCACTGA-3 0 and NeoA 5 0 -CGGCCGGAGAACCTGCGTGCAA-3 0 . ", "section_name": "Methods", "section_num": null } ]	[ { "section_content": "Acknowledgements S.D.T., L.K., O.M., L.B. and L.L. are members of the European Research Initiative for ALK-Related Malignancies (www.erialcl.net).S.D.T. receives funding from Bloodwise.T.I.M. was in receipt of a Gordon Piller PhD studentship from Leukaemia and Lymphoma Research at the time of the study.C.J.F. is supported with PhD funding from the bbsrc.S.D.T., S.M., I.A. and T.I.M. are supported with funding from the University of Ha'il, Kingdom of Saudi Arabia.Support to the Macintyre Laboratory includes the French Institut National de Cancer (INCa) PAIR Lymphoma T-COG (N°2008-021) and RT-07 Immature T/My leukemia 'Recherche Translationnelle' programmes and MD/ PhD grant funding to A.T., the Enfants et Sante ´and Socie ´te ´Franc¸aise de Cancers de l'Enfant (SFCE) and the Association Laurette Fugain (ALF2012-09).Support to the Lamant Laboratory includes INCa PAIR Lymphoma T-COG.This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub.We are grateful to Dr Phil G. Stevenson, Department of Pathology, University of Cambridge, UK for providing MHV68 for our studies, Professor Jean Soulier for CGH array analysis of human ALCL samples, Michaela Schlederer for her excellent technical assistance and Mohamed Belhocine for bioinformatics assistance. ", "section_name": "", "section_num": "" }, { "section_content": "Histology.Mice displaying clinical signs were euthanized and post-mortem performed examining all tissues for tumour formation.Tumours and/or associated tissues were fixed in 10% formalin, and subsequently paraffin embedded, sectioned and stained with haematoxylin and eosin for histological examination.All tissue processing was carried out at either the Department of Pathology, University of Cambridge, UK, or the Medical University of Vienna, Austria and sections were examined independently by at least two histopathologists. Immunohistochemistry. Immunohistochemistry was carried out with the following antibodies: CD30 (Monoclonal Mouse Anti-Human; Clone Ber-H2, 1:50, Dako, Denmark); TCR b-F1 (Monoclonal Mouse Anti-Human, Clone 8A3, 1:100, Thermo Scientific, IL 61105 USA); ALK1 mAb (1:100 kindly provided by Dr Karen Pulford, University of Oxford) as described in ref. 42. Cell lines.The human cell lines SUDHL-1, Karpas-299, HPB-ALL, DND41 and Jurkat were purchased from the DSMZ and were cultured in RPMI1640 supplemented with 10% FCS.The COST and PIO ALCL cell lines were developed in the Lamant laboratory and were grown in ISCOVE medium (Invitrogen, Cergy Pontoise, France) supplemented with 10% FCS.All the cell lines were subject to quarterly mycoplasma testing and are not on the ICLAC and NCBI Biosample misidentified cell list. Flow cytometry.Single-cell suspensions were obtained from isolated tissue samples.Cells were washed, counted and stained with the following murine phycoerythrin, fluorescein isothiocyanate, allophycocyanin or cyanine 7 (Cy7)-conjugated antibodies: CD4, CD8, CD44, CD25, CD30, CD117, CD3, Va2 TCR, Notch1 (BD Biosciences, Oxford, UK).After staining (30 min at 4 °C, 1:1,000), cells were washed and analysed using fluorescence-activated cell sorter (FACS) Canto or Fortessa flow cytometers (BD Biosciences).For intracellular stain, the cells were fixed in 0.01% formaldehyde for 10 min, permeabilized in 0.5% Tween 20 v/v in PBS (15 min in the dark at RT), cells were washed in PBS 0.1% Triton and re-suspended in 100 ml of 0.1% Triton in PBS, cells were stained and analysed as previously described.Alternatively, cell populations were sorted using a FACS Aria machine as detailed in ref. 11. RT-PCR analysis.RNA was extracted cells using an RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions and converted to cDNA using Superscript II reverse-transcriptase (RT) (Invitrogen), with dNTPs and random hexamers.PCR was carried out using primers specific to NPM-ALK 24 , RAG2 or Va2 (OTI transgene) (OT1 FWD 5 0 -ACGTGTATTCCCATCTCTGG-3 0 , OTI R 5 0 -CTGTTCATAATTGGCCCGA-3 0 ; NPM-ALK FWD 5 0 -TCCCTTG GGGGCTTTGAAATA-3 0 , NPM-ALK RVS 5 0 -CGAGGTGCGGAGCTTGCT CAG-3 0 ; RAG A 5 0 -GGGAGGACACTCACTTGCCAGTA-3 0 , RAG B 5 0 -AGTCA GGAGTCTCCATCTCACTGA-3 0 and NeoA 5 0 -CGGCCGGAGAACCTGCG TGCAA-3 0 ). Murine TCR rearrangement analysis.DNA was extracted from 5 Â 10 6 murine tumour cells or thymocytes using the DNeasy Blood and Tissue Kit (Qiagen).PCR was performed as previously described 13,43 .Briefly Vb2 (5 0 -GTAGGCACCTGTG GGGAAGAAACT-3 0 ) or Db2 forward (5 0 -GGGTCACTGATACGGAGCTG-3 0 ) primers and Jb2 reverse (5 0 -TGAGAGCTGTCTCCTACTATCGATT-3 0 ) primer (for the V-J and D-J reactions, respectively) were used to amplify rearranged TCRb regions, which were then separated by electrophoresis.Full uncropped images are shown in Supplementary Figs 11-14 for all PCR data presented. RQ-PCR of RAG status.RAG expression transcript Ct values were normalized for RNA quality and quantity relative to Abl as previously described 35 .One microgram of total RNA was reverse transcribed as described 39 and RNA quality and quantity was assessed relative to the ABL housekeeping gene, on an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA, USA).Primers used for the analysis of Rag1 were: RAG1 sense, 5 0 -AGCCTGCTGAGCAAGGTACC-3 0 ; antisense, 5 0 -GAACTGAGTCCCAAGGTGGG-3 0 ; probe, Fam-5 0 -AGCCAGCATGGCAGC CTCTTTCC-3 0 -Tamra Immunohistochemistry. Double-staining immunohistochemistry was performed using the automated Ventana Benchmark platform (Ventana Medical Systems Tuscon, AZ, USA) by sequential staining for both antibodies (first TCR b-F1, then CD30).Pre-treatment was carried out with enzyme I (Leica AR9551) for TCR b-F1 and Epitope Retrieval 1 (Leica AR9961) for CD30.Staining was developed using the Bond Polymer Refine detection kit (DAB for TCR b-F1) and Bond Polymer Refine Red detection kit (AP Fast Red for CD30) according to the manufacturer's recommendations.ALK1 staining was performed using a standard protocol.HIER procedure was performed for antigen retrieval using Target Retrieval Solution (DAKO S2369).Development was carried out using the IDetect Super Stain System HRP (ID Laboratories, IDSTM007).The signal was visualized with 3-amino-9-ethylcarbazole (ID Laboratories, BP1108). Irradiation and intravenous injection of thymocytes.Before injection, wild-type recipient mice were exposed to two rounds of irradiation to a total of 11 Grays to clear the host bone marrow.Thymocytes were obtained from the thymus of a CD4NA mouse that showed signs of dysplasia as assessed by cell surface protein expression.CD44 hi and CD44 lo populations were sorted into PBS using a FACS Aria (BD).Sorted thymocytes from CD45.2 donor BL/6 mice (3 Â 10 5 ) were then mixed with donor CD45.1 wild-type bone marrow cells (6.6 Â 10 4 ) and intravenously injected into irradiated CD45.2 recipients. Luciferase assay.In each transfection reaction 1 Â 10 7 Jurkat cells were mixed with plasmid DNA in a 0.4 cm electrode gap Gene Pulser cuvette (40 mg of pGL2-CD44-firefly luciferase, 2 mg of pRL-TK vector, and varying amounts of an NPM-ALK vector as indicated) and electroporation performed on the Gene Pulser II (Bio-Rad), at 0.25 kV and 950 mF for B25 ms.After 48 h in culture, firefly and renilla luciferase were exposed to their relevant substrates using a Stop and Glo kit (Promega), and the output measured on the Victor 3 multi-label counter (Perkin Elmer, Waltham, MA) as described previously 16 . In vivo EdU analysis of cellular proliferation.Mice were injected intraperitoneally with 300 ml of a 10 mM EdU solution in PBS.After 3 h, mice were killed and the thymus of each mouse was isolated before FACS staining for T cell markers as described in the main text.After staining (30 min at 4 °C), cells were washed and then fixed, permeabilized and EdU incorporation detected using a Click-iT EdU Flow Cytometry Assay Kit (Invitrogen).The amount of EdU incorporated was then measured by Flow Cytometry. Statistics.Data were analysed using a two-tailed Student's t-test (assuming equal variance) or log-rank test, as indicated using GraphPad Prism 6 software (La Jolla, CA). Author contributions Accession codes: The aCGH data have been deposited in the NCBI GEO database under accession code GSE67131. Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications Competing financial interests: The authors declare no competing financial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/ ", "section_name": "Additional information", "section_num": null }, { "section_content": "Histology.Mice displaying clinical signs were euthanized and post-mortem performed examining all tissues for tumour formation.Tumours and/or associated tissues were fixed in 10% formalin, and subsequently paraffin embedded, sectioned and stained with haematoxylin and eosin for histological examination.All tissue processing was carried out at either the Department of Pathology, University of Cambridge, UK, or the Medical University of Vienna, Austria and sections were examined independently by at least two histopathologists. Immunohistochemistry. Immunohistochemistry was carried out with the following antibodies: CD30 (Monoclonal Mouse Anti-Human; Clone Ber-H2, 1:50, Dako, Denmark); TCR b-F1 (Monoclonal Mouse Anti-Human, Clone 8A3, 1:100, Thermo Scientific, IL 61105 USA); ALK1 mAb (1:100 kindly provided by Dr Karen Pulford, University of Oxford) as described in ref. 42. Cell lines.The human cell lines SUDHL-1, Karpas-299, HPB-ALL, DND41 and Jurkat were purchased from the DSMZ and were cultured in RPMI1640 supplemented with 10% FCS.The COST and PIO ALCL cell lines were developed in the Lamant laboratory and were grown in ISCOVE medium (Invitrogen, Cergy Pontoise, France) supplemented with 10% FCS.All the cell lines were subject to quarterly mycoplasma testing and are not on the ICLAC and NCBI Biosample misidentified cell list. Flow cytometry.Single-cell suspensions were obtained from isolated tissue samples.Cells were washed, counted and stained with the following murine phycoerythrin, fluorescein isothiocyanate, allophycocyanin or cyanine 7 (Cy7)-conjugated antibodies: CD4, CD8, CD44, CD25, CD30, CD117, CD3, Va2 TCR, Notch1 (BD Biosciences, Oxford, UK).After staining (30 min at 4 °C, 1:1,000), cells were washed and analysed using fluorescence-activated cell sorter (FACS) Canto or Fortessa flow cytometers (BD Biosciences).For intracellular stain, the cells were fixed in 0.01% formaldehyde for 10 min, permeabilized in 0.5% Tween 20 v/v in PBS (15 min in the dark at RT), cells were washed in PBS 0.1% Triton and re-suspended in 100 ml of 0.1% Triton in PBS, cells were stained and analysed as previously described.Alternatively, cell populations were sorted using a FACS Aria machine as detailed in ref. 11. RT-PCR analysis.RNA was extracted cells using an RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions and converted to cDNA using Superscript II reverse-transcriptase (RT) (Invitrogen), with dNTPs and random hexamers.PCR was carried out using primers specific to NPM-ALK 24 , RAG2 or Va2 (OTI transgene) (OT1 FWD 5 0 -ACGTGTATTCCCATCTCTGG-3 0 , OTI R 5 0 -CTGTTCATAATTGGCCCGA-3 0 ; NPM-ALK FWD 5 0 -TCCCTTG GGGGCTTTGAAATA-3 0 , NPM-ALK RVS 5 0 -CGAGGTGCGGAGCTTGCT CAG-3 0 ; RAG A 5 0 -GGGAGGACACTCACTTGCCAGTA-3 0 , RAG B 5 0 -AGTCA GGAGTCTCCATCTCACTGA-3 0 and NeoA 5 0 -CGGCCGGAGAACCTGCG TGCAA-3 0 ). Murine TCR rearrangement analysis.DNA was extracted from 5 Â 10 6 murine tumour cells or thymocytes using the DNeasy Blood and Tissue Kit (Qiagen).PCR was performed as previously described 13,43 .Briefly Vb2 (5 0 -GTAGGCACCTGTG GGGAAGAAACT-3 0 ) or Db2 forward (5 0 -GGGTCACTGATACGGAGCTG-3 0 ) primers and Jb2 reverse (5 0 -TGAGAGCTGTCTCCTACTATCGATT-3 0 ) primer (for the V-J and D-J reactions, respectively) were used to amplify rearranged TCRb regions, which were then separated by electrophoresis.Full uncropped images are shown in Supplementary Figs 11-14 for all PCR data presented. RQ-PCR of RAG status.RAG expression transcript Ct values were normalized for RNA quality and quantity relative to Abl as previously described 35 .One microgram of total RNA was reverse transcribed as described 39 and RNA quality and quantity was assessed relative to the ABL housekeeping gene, on an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA, USA).Primers used for the analysis of Rag1 were: RAG1 sense, 5 0 -AGCCTGCTGAGCAAGGTACC-3 0 ; antisense, 5 0 -GAACTGAGTCCCAAGGTGGG-3 0 ; probe, Fam-5 0 -AGCCAGCATGGCAGC CTCTTTCC-3 0 -Tamra Immunohistochemistry. Double-staining immunohistochemistry was performed using the automated Ventana Benchmark platform (Ventana Medical Systems Tuscon, AZ, USA) by sequential staining for both antibodies (first TCR b-F1, then CD30).Pre-treatment was carried out with enzyme I (Leica AR9551) for TCR b-F1 and Epitope Retrieval 1 (Leica AR9961) for CD30.Staining was developed using the Bond Polymer Refine detection kit (DAB for TCR b-F1) and Bond Polymer Refine Red detection kit (AP Fast Red for CD30) according to the manufacturer's recommendations.ALK1 staining was performed using a standard protocol.HIER procedure was performed for antigen retrieval using Target Retrieval Solution (DAKO S2369).Development was carried out using the IDetect Super Stain System HRP (ID Laboratories, IDSTM007).The signal was visualized with 3-amino-9-ethylcarbazole (ID Laboratories, BP1108). Irradiation and intravenous injection of thymocytes.Before injection, wild-type recipient mice were exposed to two rounds of irradiation to a total of 11 Grays to clear the host bone marrow.Thymocytes were obtained from the thymus of a CD4NA mouse that showed signs of dysplasia as assessed by cell surface protein expression.CD44 hi and CD44 lo populations were sorted into PBS using a FACS Aria (BD).Sorted thymocytes from CD45.2 donor BL/6 mice (3 Â 10 5 ) were then mixed with donor CD45.1 wild-type bone marrow cells (6.6 Â 10 4 ) and intravenously injected into irradiated CD45.2 recipients. Luciferase assay.In each transfection reaction 1 Â 10 7 Jurkat cells were mixed with plasmid DNA in a 0.4 cm electrode gap Gene Pulser cuvette (40 mg of pGL2-CD44-firefly luciferase, 2 mg of pRL-TK vector, and varying amounts of an NPM-ALK vector as indicated) and electroporation performed on the Gene Pulser II (Bio-Rad), at 0.25 kV and 950 mF for B25 ms.After 48 h in culture, firefly and renilla luciferase were exposed to their relevant substrates using a Stop and Glo kit (Promega), and the output measured on the Victor 3 multi-label counter (Perkin Elmer, Waltham, MA) as described previously 16 . In vivo EdU analysis of cellular proliferation.Mice were injected intraperitoneally with 300 ml of a 10 mM EdU solution in PBS.After 3 h, mice were killed and the thymus of each mouse was isolated before FACS staining for T cell markers as described in the main text.After staining (30 min at 4 °C), cells were washed and then fixed, permeabilized and EdU incorporation detected using a Click-iT EdU Flow Cytometry Assay Kit (Invitrogen).The amount of EdU incorporated was then measured by Flow Cytometry. Statistics.Data were analysed using a two-tailed Student's t-test (assuming equal variance) or log-rank test, as indicated using GraphPad Prism 6 software (La Jolla, CA). Author contributions ", "section_name": "", "section_num": "" }, { "section_content": "Accession codes: The aCGH data have been deposited in the NCBI GEO database under accession code GSE67131. Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications Competing financial interests: The authors declare no competing financial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/ ", "section_name": "Additional information", "section_num": null } ]
10.1038/ncomms11394	Epidermal Notch1 recruits RORγ+ group 3 innate lymphoid cells to orchestrate normal skin repair	<jats:title>Abstract</jats:title><jats:p>Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ<jats:sup>+</jats:sup> ILC3s into wounded dermis; RORγ<jats:sup>+</jats:sup> ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ<jats:sup>+</jats:sup> ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair.</jats:p>	[ { "section_content": "ound healing in epithelial tissues involves three phases; inflammation, proliferation and remodelling 1,2 .Following injury, neutrophils and phagocytic cells infiltrate clearing microbial contaminants.The subsequent proliferative phase is characterized by epidermal proliferation, extracellular matrix deposition, granulation, vascularization and wound contraction.Following wound closure, excess cells and debris are removed in the remodelling phase and the extracellular matrix is reorganized to restore tissue strength.At the wound site, adaptive and innate immune cells regulate the skin wound healing process through production of cytokines, antimicrobial peptides and growth factors.Wound healing is not obligate on a functional immune system; most immune-deficient models heal wounds 3,4 .However, research shows macrophages have key roles in wound closure 5 .Further in mice, injury activates epidermalresident gd T cells called dendritic epidermal T cells (DETCs) to produce growth factors and inflammatory cytokines that control the epithelial injury response 6 .Whether the immune system helps or hinders effective repair remains controversial.Athymic nude mice undergo complete, scarless repair 3,7 , while conversely, macrophage persistence in wound sites leads to fibrosis and scar formation; thus proper timing of immune cell entry and exit is critical for normal repair 1,8 . The Notch pathway is a key, cell-autonomous signalling pathway that directs cell fate and has pleiotropic functions in the skin [9][10][11] .Notch signalling initiates when a Notch ligand binds to one of the four receptors present on mammalian cells, which causes receptor cleavage and enables the intracellular domain to undergo nuclear translocation, and effect changes in gene transcription 12 .Expression of all four Notch receptors in the epidermis has been reported 13 .However, genetic studies suggest Notch1 and Notch2 are the primary receptors needed to regulate the cell differentiation required to maintain hair and skin epithelia 14 .A previous study using topically-applied pan-Notch activators and inhibitors suggested that Notch might be involved in wound healing 15 , and our previous work showed that forced, ectopic epidermal Notch1 activity resulted in extensive epidermal proliferation and severe inflammation, two phenotypic hallmarks of skin wound healing 9,16,17 . Innate lymphoid cells (ILCs) are rare populations of lymphocytes that have key roles in secondary lymphoid tissue formation, homoeostasis and rapid production of cytokines in response to pathogen infection.ILCs are classified by cytokine production and expression of transcription factors into three groups, termed group 1, 2 or 3, and are found largely within the stroma of mucosal tissues 18 .Group 1 or ILC1s in mucosal epithelium produce interferon-g-mediated responses against pathogens and are thought to contribute to intestinal pathologies when dysregulated, while Group 2 (ILC2s) are linked to allergenic responses and, in skin, to atopic dermatitis through the production of type 2 cytokines, IL5 and IL13 (refs 19-23).Group 3 or ILC3s are characterized by the expression of RORg transcription factor and have key roles in the maintenance and repair of epithelial tissues.Further, ILC3s contribute to intestinal epithelial repair through IL22 upregulation 24 , and through ILC3-mediated release of granulocyte macrophage colony stimulating factor (GM-CSF) controlling macrophage and dendritic cell responses to gut commensal microflora 25,26 .In skin, recent studies have linked ILC3s to the pathogenesis of psoriasis through IL23a stimulated IL17 and IL22 production.However, despite the similarity of skin and intestine as barrier organs, the contribution of ILCs to the physiology of normal skin tissue repair remained unstudied. In this study, we show that damage activates epidermal Notch signalling.Notch in turn, controls dermal entry of inflammatory cell subsets, including NKp46 low/-CD4 þ ILC3s to wound sites.In uninjured mouse and human skin, dermis-resident ILC3s are exceptionally rare, but Notchcontrolled epidermal signals recruit ILC3s in a TNFa-dependent process.Furthermore, using RORg þ -deficient mice, we present evidence that RORg þ ILC3s, in wounds, produce IL17F and CCL3 (also known as MIP1a) and have key roles the normal healing response; ILC3s control epidermal proliferation and macrophage entry into the dermis. ", "section_name": "W", "section_num": null }, { "section_content": "Skin injury activates Notch1 signalling.To determine the roles of Notch in wound healing, 6-week-old mice were given two, full-thickness punch wounds, and back skin tissues were analysed for Notch1 at 1, 3/4 and 7 days post wounding (dpw).Wounding caused an immediate increase (B7 Â ) in Notch1 activity 1 dpw with peak activity detected 4 dpw (B16 Â ) (Fig. 1a).Stained sectioned tissues revealed Notch1 was primarily active in suprabasal epidermal keratinocytes 1 and 4 dpw (Fig. 1b and Supplementary Fig. 1).A significant increase in Jagged1 and Jagged2 messenger RNA (mRNA) expression was detected by quantitative real time PCR (QPCR) within 8 h of injury (Fig. 1c).Published studies show skin dendritic cells in the mouse ear can upregulate the Notch ligands Jagged1 and Jagged2 in response to damage 27 , however we detected Jagged1 protein in basal and suprabasal epidermal layers 2 and 5 dpw.This suggests that Notch ligands are available locally in the epidermis to activate Notch during wound healing (Fig. 1d).We conclude that the Notch pathway is active in the inflammatory and proliferation phases of wound healing. Epidermal Notch1 drives dermal immune responses after injury.Our previous work showed that epidermal Notch activity, via Jagged1, stimulates epidermal production of pro-inflammatory TNFa and persistent Notch activity leads to CD3 þ CD4 þ T cell recruitment 9,17 .Thus we hypothesized that Notch signalling leads to inflammatory cell recruitment following injury (Fig. 2a).We quantified populations of dermal immune cells in unwounded skin (UW) and punch-wounded skin during the inflammatory, proliferation and early remodelling phases at 2, 5 and 8 dpw, respectively.Wounding induced the expected early influx of CD11b þ Gr-1 þ polymorphonuclear neutrophils and CD11b þ F4/80 þ monocytes/macrophages (Supplementary Fig. 2a,b).The percentage of anti-inflammatory M2 macrophages was determined by CD206 marker expression; in alignment with previously published studies, the macrophage population was heterogeneous throughout skin repair, with 78-87% of macrophages expressing the M2 marker at all wound stages investigated (Supplementary Fig. 2d) 28 .CD206 marker expression was quantified on spleen cells as an experimental control and, in agreement with published studies, we detected B20% of splenocytes expressing CD206 (Supplementary Fig. 2d).NK1.1 þ (NK and/or NKT) cell numbers peaked at 8 dpw (Supplementary Fig. 2c) consistent with a role in tissue remodelling.Next, we examined the innate lymphoid cell populations, as these cells were known to have roles in intestinal tissue repair and skin pathology.Increased numbers of ILC2s were detected by 8 dpw; while an increase in ILC1s was detected at 5 and 8 dpw during the late proliferation and remodelling phases of wound healing when IFNg levels are high (Supplementary Fig. 2e,f) 19 .RORg þ CD127 þ Lin -ILC3s were detected, yet these cells comprised a very small proportion of total back skin cells, o1% of total dermal cells, at all time points (Fig. 2b,c).In summary, our data show that injury controls localized and temporal recruitment of leukocytes including ILC subsets. We theorized that the small numbers of ILC3s detected by flow cytometry were likely due to the inclusion of dermal cells from both wounded and non-wounded skin.To test if wounding caused localized recruitment of ILC3s to wound sites, we examined the distribution of RORg þ ILC3s in sectioned wound tissues (Fig. 2d).At all time points post wounding ILC3s were detected in the dermis.At 2 dpw, most ILC3s were found in the reticular dermis, however by 5 and 8 dpw ILC3s were detected in both the reticular and papillary dermis, suggesting active recruitment of cells from the reticular blood vascular network into the dermis (Fig. 2d).Similarly in human skin, we detected infiltration of dermal RORg þ CD127 þ CD3 -ILCs following wounding using a punch biopsy (Supplementary Fig. 3a).In mice tissues, the distribution of ILC3s was quantified relative to the epidermal wound edge as defined by the distinctive phenotypic appearance of this tissue (Fig. 2e,f).Most RORg þ ILC3s were localized nearest to the wound site within 1 mm of the wound edge (zone 1) and maximal numbers were detected at 5 dpw during the proliferative phase (Fig. 2d).As controls, sections from wounded RORg À / À mice were stained with RORg antibodies; no staining was detected confirming specific immunoreactivity of RORg antibodies in tissue sections (Supplementary Fig. 4a).ILC3 subsets differentially express NKp46 and CD4, so we analysed the surface marker expression in RORg eGFP/ þ skin (Supplementary Fig. 4b) 29 .The majority of RORg-eGFP þ cells were CD4 þ , and expressed very low levels of NKp46 with heterogeneous expression of CD117, thus CD4 þ NKp46 low/-ILC3s.Further, these ILC3s expressed the chemokine receptors, CCR6, CXCR4 and CXCR5, important for ILC3 chemotaxis (Supplementary Fig. 4b). Having established the timed cascade of immune cell infiltration in skin wounds, we next investigated the link between injury-activated epidermal Notch and the dermal accumulation of ILC3s and other immune cell subsets.Previously we published a K14NICDER transgenic mouse model where Notch1 activity can be tightly controlled in the epidermis by drug application.A full characterization of this mouse model has been previously published 9,17 .In brief, the K14NICDER transgene contains a truncated, Notch1 intracellular domain (NICD) that can be temporally and spatially activated by 4-hydroxy-tamoxifen (4OHT) in the basal, keratin 14-expressing epidermis.Here, Notch was activated by 4OHT in transgenic mice for 3 days.Control mice were likewise 4OHT treated and sectioned tissues were antibody stained to visualize Gr-1 þ neutrophils, F4/80 þ macrophages and RORg þ ILC3s (Fig. 3a-c).Only rare neutrophils were detected in both 4OHT-treated transgenics and controls (Fig. 3a), but a substantial increase in the number of macrophages and ILC3s were detected in transgenic dermis compared to control tissue (Fig. 3b,c), suggesting that epidermal Notch activation in the absence of tissue damage is sufficient to drive recruitment of subsets of wound-induced dermal immune cells. Next, we inhibited Notch1 receptor activation using NRR1 blocking antibodies, which block ligand binding to Notch1 receptor but not the other Notch receptors, thus specifically inhibiting Notch1 signalling 30 .As controls, mice were treated with NRR2 blocking antibodies, which have equivalent efficacy against Notch2 receptors, isotype IgG antibodies or phosphate buffered saline (PBS).Mice were injected intraperitoneally (i.p.) for 7 days before wounding, and we confirmed that antibody treatment caused expected systemic effects; NRR1-treated mice were blocked in thymocyte development and NRR2-treated mice were blocked in marginal zone B cell maturation, Notch1 and Notch2 dependent processes, respectively (Supplementary Fig. 5a) 30 .Following wounding, closure rates were measured and tissues collected 2 or 5 dpw.Wounds treated with the Notch1 blocking antibody, NRR1, were significantly more open (77%) 2 dpw compared to mice injected isotype IgG control (66%), but by 5 dpw wound size in NRR1-treated mice did not differ from controls (Fig. 4a,b).NRR1-treated wounds displayed a distinct phenotype; wounds showed increased erythema, with domed margins (signs of abnormal wound granulation), compared to controls (Fig. 4a).Following treatment with NRR1, detectable nuclear activated Notch1 was reduced in the epidermal layers as detected by antibody staining, and total cleaved, active Notch1 protein was reduced in whole skin preparations quantified by western immunoblotting (Supplementary Fig. 5c,d).No difference in basal and suprabasal marker expression was detected in NRR1 and control-treated skin suggesting NRR1 treatment did not block differentiation of epidermal keratinocytes (Supplementary Fig. 5e). The impact of reduced Notch1 activation on wound-recruited dermal immune cells was assessed in sectioned tissues and by flow cytometry in NRR1-treated mice.At 2 dpw, most dermal immune cell populations were similar in NRR1 and IgG control-treated wounds (Fig. 4c; Supplementary Fig. 5f) with the exception of ILC3s; fewer dermal ILC3s were detected adjacent to wounds at 2 dpw (Supplementary Fig. 4f).At 5 dpw, reduced Notch1 levels led to fewer dermal CD11b þ leukocytes, NK1.1 þ cells, ILC1s and ILC3s (Fig. 4c,d).We confirmed that Notch1, but not Notch2 activity was directly linked to RORg þ ILC3 recruitment; ILC3 recruitment was inhibited in wounded mice treated with NRR1, but not NRR2, blocking antibodies (Fig. 4e). Taken together these results demonstrate that Notch, as a damage response factor, directs recruitment of dermal immune cells.Further, we found a novel link between epidermal Notch activity and ILC3 recruitment to wound sites.Given the key roles of ILC3s in regulating mucosal tissue repair, we next investigated the Notch-regulated mechanism leading to ILC3 recruitment and their functions in skin repair. Epidermal Notch1 drives TNFa-mediated recruitment of ILC3s.To investigate the mechanism by which Notch1 leads to ILC3 recruitment, dermal and epidermal gene expression profiles of uninjured, K14NICDER transgenic mouse back skin (NIH GEO:GSE23782 (ref.9)) were examined.IPA software was used to identify secreted factors upregulated in 4OHT-treated K14NICDER skin (6 Â or greater) that could facilitate immune cell infiltration into the dermis and the subsequent activation (Fig. 5a,b).Analysis identified several key candidates that could regulate innate immune cell recruitment and activation (NIH GEO: GSE29777 (ref.31)).Of these, Notch-regulated factors CCL20 (14.1 Â ), CXCL12 (8.0 Â ) and CXCL13 (6.5 Â ) were of particular interest as these factors directly regulate ILC3s; and we showed that the cognate receptors CCR6, CXCR4 and CXCR5 for the ligands CCL20, CXCL12 and CXCL13, respectively, are expressed on wound-recruited ILC3s (Supplementary Fig. 4b and Fig. 5a,b) 32 .Further, TNFa (17.1 Â ) was also of interest, as it can direct CCL20 expression in the epidermis 33 and CCL20 is a key recruitment factor for RORg þ ILC3s in lymphoid tissues 34 . The ligand/receptor pairings of CCL20/CCR6, CXCL12/ CXCR4 and CXCL13/CXCR5 have vital chemotactic functions in many immune cells including ILCs, dendritic cells (DCs) and T and B cells.Innate immune cell recruitment in mucosal epithelium is CCL20/CCR6 dependent 35 .Skin wounding caused robust, early activation of Notch1 (Fig. 1b), thus we measured CCL20, CXCL12 and CXCL13 mRNA levels at early time points (4, 8, 16, 24, 40, 48 and 120 h) after injury by quantitative PCR (qPCR) (Fig. 5c).CCL20 and CXCL13 levels were elevated immediately following wounding and returned to near background levels by 40 h post injury and remained low (Fig. 5c).No increase in CXCL12 mRNA levels was detected from 0 to 24 h post wounding, with a moderate increase in CXCL12 levels after 48 h.In human wounds, CXCL13 and CCL20 were also differentially regulated with highest expression observed early during the inflammatory phase (Supplementary Fig. 3b). The early activation of CCL20 and CXCL13 in wounded tissues, and in Notch-active transgenic mice, suggests that Notch has a role in their activity.Mice with reduced Notch activity (NRR1 treated) had lower levels of CXCL13, but not CCL20 compared to controls (Fig. 5d).These data link Notch activity to CXCL13 activation, yet we cannot rule out a role for Notch1 in CCL20 activation as in our model Notch activity is reduced, not fully blocked.However, CXCL13 and CCL20 were transcriptionally downregulated within 24-h of injury, while Notch1 activity remained high (Fig. 1a) suggesting other transcriptional regulators are also likely important.To test whether CCL20 and CXCL13 are essential chemokines in ILC3 recruitment into skin wounds, we examined ILC3 recruitment in mutant mice deleted for single chemotactic pathways.CCR6 À / À and CXCL13 À / À mice and their respective littermate controls were punch wounded; wound closure rates were monitored and tissue sections were analysed by immunodetection for RORg þ ILC3 recruitment at 5 dpw.There was no detectable difference in wound closure rates between the mutant and control mice.The number of dermal ILC3s in antibodystained CXCL13 À / À mutant tissue sections was not significantly different from controls (Supplementary Fig. 6a,b), whereas, a small increase in ILC3 numbers was detected in CCR6 À / À mutants compared to controls (Supplementary Fig. 6c,d).We identified injury-induced upregulation of a third chemokine in human wounds, CXCL16, that is known to regulate the chemotaxis of both mouse ILC3s and human innate immune cells (Supplementary Fig. 3b) 29,36 .Taken together these results suggest multiple, redundant pathways are important in ILC3 chemotaxis. Lastly, we found levels of TNFa corresponded to levels of Notch activity in skin with increased (K14NICDER) and decreased Notch activity following injury (wounded NRR1treated mice) (Fig. 5a,d) 9 .Thus, we determined if TNFa downstream of epidermal Notch activity mediates ILC3 localization.In unwounded K14NICDER, TNFa activity was blocked by injecting a TNFa blocking antibody, or PBS control, daily for 7 days before 4OHT-activation of Notch.Antibody staining on tissue sections revealed that anti-TNFa treatment blocked Notch-mediated ILC3 localization into the skin (Fig. 5e). To confirm that TNFa regulates ILC3 dermal localization in wounds, wounded animals were treated with recombinant TNFa, TNFa blocking antibody or PBS daily.By 5 2dpw, a substantial difference was observed between the three groups of mice: TNFa accelerated wound closure and increased ILC3 numbers (1.8 Â ), while the antagonist inhibited closure with reduced numbers of ILC3s (0.4 Â ) (Fig. 5f,g and Supplementary Fig. 6e).Mechanistically, TNFa bioavailability may impact ILC3 recruitment through regulating CCL20.Previous studies have shown that CCL20 is produced from epidermal keratinocytes in response to TNFa stimulation 33 , and we confirmed that primary dermal cells respond similarly (Supplementary Fig. 6f).However, it is almost certain that multiple factors control CCL20 expression; TNFa alone or in combination with IL-1b, CD40 ligand, IFNg, and IL17 can all stimulate CCL20 production in epidermal keratinocytes and dermal fibroblasts (Supplementary Fig. 6f).In summary, these results implicate a Notch/TNFa mechanism, working in conjunction with chemokines, in ILC3 recruitment into skin wounds. The functional role of Notch-recruited ILC3s in skin wounds. Innate lymphocytes and other cell types, including Th17 cells, DETCs and dermal gd T cells, can be potent sources of IL17 and IL22 cytokines that attenuate immune responses 18,[38][39][40] .IL17A production drives psoriatic inflammation in skin 41 and imiquimod-induced psoriatic-like lesions in mice express high levels of IL17A and IL17F 42 .Further, the IL22 receptor signalling pathway has a key role in skin wound healing 43 and IL22 has a key role in thymus regeneration 20 and accelerates keratinocyte closure in in vitro wound scratch assays 24 .ILC3s isolated from 5 dpw dermis were analysed for cytokine production by intracellular flow cytometry analysis (Fig. 6a).We analysed IL17A, IL17F and IL22 cytokine production in unstimulated and phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced cells.Although, we detected low levels of IL22-, IL17A-and IL17Fproducing cells among the population of unstimulated ILC3s, following PMA/ionomycin, the majority of ILC3s predominantly expressed IL17F, with a small fraction of cells double positive for IL17A and IL17F.Similarly, human wounds populated with dermal ILC3s have elevated levels of IL17F and IL17A (Supplementary Fig. 3c).IL22 is difficult to detect by flow cytometry, so in addition we examined mRNA levels by qRT-PCR.We detected increased IL22 levels by 2 dpw consistent with a potential role for ILC3s in producing IL22 needed for skin repair (Fig. 6b).In summary, results suggest that ILC3s contribute to normal physiological tissue repair in the skin through production of IL17F and to a lesser extent IL17A and IL22. To investigate the function of Notch-recruited ILC3s in skin tissue repair, we examined the wound healing in RORg À / À and littermate controls (Fig. 7a).We developed non-invasive 3D imaging methods to quantify wound closure rates in these animals.Briefly, mice were photographed repeatedly using a 3D camera and an accurate measurement of the wound perimeter was calculated over time (Fig. 7b and Supplementary Fig. 7a-e).Wounds were significantly more open in RORg À / À mice given 4 mm punch wounds compared to control littermates at 5 dpw (Fig. 7a,b and Supplementary Fig. 6e).However, by 8 dpw, wounds were of a similar openness in mutant and control mice with re-epithelialization complete in both (Fig. 7b and Supplementary Fig. 7e).Wound-healing defects were exacerbated in RORg À / À mice with larger diameter (6mm) wounds.Larger wounds take longer to close and wound were significantly more open at 8dpw in RORg À / À mice receiving large wounds (Supplementary Fig. 7f).Delayed wound closure in RORg À / À mice with small wounds (4mm) was linked to delayed re-epithelialization of the keratinocyte layer; at 5 dpw, RORg À / À mice had a marked reduction in Ki67 þ epidermal cells compared to controls (Supplementary Fig. 8a-c).We confirmed that phenotypes observed in the RORg À / À mice resulted from loss of RORg þ ILC3s.We transplanted spleen cells from Rag2 À / À mice (lacking ab and gd T cells, but containing RORg þ ILCs) into RORg À / À mice 24 h before wounding.Cell transplantation ameliorated wound pathology observed in control RORg À / À mice and donor RORg þ ILCs could be detected adjacent to wound sites (Supplementary Fig. 8d).Therefore, loss of ILCs in RORg À / À mice directly causes wound pathology. Crosstalk between ILC3s and macrophages has been shown to regulate macrophage function in intestinal homoeostasis 26 , thus we examined the inflammatory infiltrate in wounded RORg À / À mice.We detected no change in neutrophil, intra-epidermal macrophage and ILC2 numbers (Fig. 7c and Supplementary Fig. 8e,f), however, a threefold reduction in F4/80 þ dermal macrophages was detected 2 dpw, as quantified in sectioned tissues (Fig. 7c, bottom right panel), compared to wildtype littermates.Rag2 À / À controls, which lack ab and gd T cells but contain ILC3s, had normal macrophage numbers.Anti-Thy1.2antibodies were used to deplete ILCs in the Rag2 À / À background and 3.6-fold fewer ILC3s, and 2.7-fold decrease in ILC2s were detected by flow cytometry in antibody-treated animals 2 dpw (Supplementary Fig. 8g,h) 44 .Anti-Thy1.2-treatedRag2 À / À mice had deficient macrophage numbers at skin wound sites confirming that delayed macrophage recruitment resulted from specific loss of dermal ILC3s, not T lymphocytes, early in the wound-healing programme.In NRR1-treated mice where ILC3 numbers are reduced, but not to zero, F4/80 þ macrophages could be detected, although few were found adjacent to the site of injury compared to controls (Fig. 7c).By 5 dpw, F4/80 þ cells were plentiful within the dermis in wildtype and RORg À / À wounds suggesting that loss of ILC3s causes a delay, but not a block in macrophage/monocyte infiltration (Fig. 7d).NRR1-treated animals had reduced numbers of F4/80 þ macrophages at 5 dpw.Quantification by flow cytometry revealed similar trends; CD11b þ cell numbers were at similar levels in both mutant and control wounds by 5 dpw, while reduced in NRR1-treated animals (Fig. 7d, bottom right panel).These results are consistent with previous reports suggesting Notch1 has additional roles in regulating macrophage/monocyte responses to tissue damage 45 . Decreased levels of CCL3, a key monocyte/macrophage recruitment chemokine, were detected in RORg À / À skin at 2 dpw compared to wildtype and Rag2 À / À controls, but not in day 5 wounds (Fig. 7e).We analysed whether ILC3s produce CCL3; CCL3 was detected in ILC3s isolated from 2 dpw, but not 5 dpw.This suggests that CCL3 production by ILC3s is required for early macrophage entry into wounded tissues in the healing programme (Fig. 7f). ", "section_name": "Results", "section_num": null }, { "section_content": "Dysregulated Notch signalling has been linked to aberrant human and mouse wound healing and our previous work linked aberrant Notch activity with human keloid formation, however, the mechanistic detail or the site of action was unknown 9,15,[46][47][48] .Here, using genetic and chemical tools to modulate Notch activity 17,30,49 , we have shown that Notch1 activity in epidermal keratinocytes has an essential temporal role in the wound-healing process through action on dermal immune cell recruitment and activation (Fig. 8).These findings are important, as it is clear that (control) and RORg À / À .(b) Quantitative analysis using 3D imaging; wound openness is measured in control and RORg À / À wounds at 5 dpw and 8 dpw. The mean openness of RORg À / À wounds is significantly greater than that of wildtype mice at 5 dpw (n correct entry and exit of immune cells is essential for conventional repair.Notch1 levels were highest in the inflammatory and proliferation phases of the wound response, and during the later remodelling phase when the epithelium has closed 2 , Notch1 activity returns to levels detected in UW.Jagged1 and Jagged2 are similarly upregulated after injury and Jagged1 protein was detected in the epidermis adjacent to wounds, suggesting that Notch is activated through classical ligand-receptor interactions in wound repair.Reducing epidermal Notch1 activity inhibits wound closure and innate immune cell recruitment, and activating Notch1 in the epidermis without tissue or barrier damage is sufficient to drive expression of TNFa, CXCL13 and CCL20 in the skin and induce recruitment of ILC3s (Fig. 8).Consistent with our findings using NRR1 blocking antibodies, wound healing is also impaired in Notch1 þ / À mice; Notch1 þ / À mice have reduced levels of TNFa and fewer F4/80 þ macrophages near the site of injury 45 .The authors conclude these defects arise from loss of Notch1 activity in macrophages and inhibition of macrophage cytokine production 45,50 .However, our results suggest that reduced Notch signalling in epithelial keratinocytes in Notch1 þ / À mice likely contributes to woundhealing defects.In our experiments, whole skin protein lysates were used and as such some cleaved Notch1 protein may come from macrophages, although the contribution is likely to be minimal.In situ antibody labelling showed strong nuclear expression of active Notch1 that was detected throughout the epidermis in the inflammatory phase.Moreover, at 7 dpw, when Notch1 levels decreased in wounds as detected by protein blotting, macrophages are still persistent within the dermis. It is well established that Notch1 has a cell-autonomous role in lineage specification of cells within both adaptive and innate immune systems 51 .Here, we demonstrate that Notch activity in non-immune cells initiates an event cascade controlling inflammation and immune regulation in the skin.Neutrophil recruitment, however, was unaffected by changes in Notch1 activity (Fig. 2a).Neutrophil recruitment is mediated by the release of danger signals or damage-associated molecular patterns, normally cytoplasmic DNA, from disrupted cells and tissues.It has been shown previously that neutrophils are recruited to sites of sterile skin injury in a leukotriene LTB4 dependent process and occurs in very short time scales, thus we believe it is completely independent of the Notchmediated recruitment of ILC3s and other innate immune cells 52,53 . Recruitment of NKp46 low/ À ILC3s into wounded dermis from the circulating blood was demonstrated by localization of dermal ILC3s after transfer of Rag2 À / À splenocytes into RORg-deficient mice.ILC3s can be detected in human blood and in psoriasis lesions; the number of ILC3s in blood increased correspondingly with the influx of dermal ILC3s 23 .Recruitment of ILCs into mucosal epithelia is a CCL20/CCR6 dependent process 54 , and we hypothesized that Notch1-mediated, wound-induced ILC3 recruitment would employ similar mechanisms.However, results from genetic deletion of the CCL20/CCR6 or CXCL13/CXCR5 signalling in CCR6 À / À and CXCL13 À / À deficient mice suggest redundancy in cell recruitment mechanisms, implicating a potential role for other chemotactic factors including CXCL16, sphingosphine-1-phosphate, prosteoglandins, retinoic acid and other chemokines all known to be expressed in the skin 55 .CXCL12 may also function as a chemotactic factor.Although we detected no change in CXCL12 gene transcription within 24 h of injury (Fig. 5c), activated platelets are a potent source of CXCL12 (ref.56).Thus, it cannot be ruled out that platelet-derived CXCL12 protein may contribute to ILC3 recruitment into skin wounds.Modulating TNFa levels had a direct consequence on the number of ILC3s at wound sites and the rate of wound healing.Moreover, Notch is a powerful inducer of TNFa and blocking TNFa activity downstream of activated Notch reduced the number of dermal ILC3s 9 . RORg þ ILC3s help facilitate skin repair through promoting epidermal re-epithelialization and monocyte/macrophage recruitment into the dermis in addition to the role of RORg þ LTi cells in lymphoid tissue formation.Using a combination of Thy1.2 depleted Rag2 À / À mice and cell transfers we have shown there is no connection between lymphoid tissue formation and ILC3 function in skin wound repair.What remains uncertain is whether ILCs have discrete roles in both these processes, or whether detected changes in epithelial proliferation are a direct consequence of delayed early macrophage influx into wounds.ILC3s secrete CCL3, an important monocyte/macrophage chemokine attractant 57 , early (2 dpw) but not later (5 dpw) in the wound repair programme.RORg À / À mice had reduced levels of CCL3 mRNA at 2 dpw compared to wildtype controls and T lymphocyte deficient Rag2 À / À mice.These data suggest simplistically that, in the absence of ILC3s, reduced CCL3 production is the cause of delayed macrophage entry.However, in reality macrophage recruitment into injured tissues is much more complex.Macrophages themselves can produce CCL3 and other chemokines, including CXCL2 (also known as MIP2) and CCL2, have key recruitment functions 58- 60 .Further, more recent work has identified that monocytes with distinct phenotypic characteristics are recruited into wound tissues at different times, suggesting an additional specificity of macrophage recruitment signalling that is yet to be fully understood 61 . In conclusion, we propose that Notch1 acts a damage ''sensor'' in epithelial tissues, transmitting a ''stress/injury signal'' and instigating tissue repair through recruitment of ILC3s and macrophages.Given the importance of ILC3s and Notch signalling in maintenance and organization of the intestinal epithelium, we propose that it is likely a Notch-mediated ''signal'' is conserved amongst epithelial tissues required to undergo periodic repair.regulates chemokine production in epidermal and dermal cells; injuryinduced Notch1 activity upregulates TNFa, which can induce CCL20 in epidermal and dermal cells (grey cells).TNFa downstream of Notch1 regulates ILC3 localization within the dermis surrounding the wound and ILC3s are key sources of IL17F and early CCL3 production in the woundhealing cascade.ILC3s subsequently influence epidermal proliferation and macrophage (Mf) recruitment, two key events in the skin wound-healing programme. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Animal models.K14NICDER (C57BL/6 Â CBA background) and RORg À / À , Rag2 À / À , CCR6 À / À and CXCL13 À / À mice in C57BL/6 background have been described previously 9,[62][63][64] .All experimental procedures were performed with ethical permission by Durham and University of York under UK government Home Office licences to CA and MC (60/3941 and 60/4178).Experiments followed the national and institutional guidelines for the care and use of animals on the basis of the Animal (Scientific Procedures) Act 1986 and where possible ARRIVE guidelines.The effect of RORg deletion on wound healing was previously uninvestigated, thus pre-specified effects prior to experimentation could not be determined and power calculations were not employed.Mice were housed in pathogen-free facilities and had access to food and water ad libitum.In K14NICDER mice, ER-inducible transgenes were activated by topical application of 2 mg of 4OHT (H6278, Sigma) dissolved in acetone. For wounding experiments, mice were anesthetized using 2% inhaled isoflurane and then injected subcutaneously with the analgesic, Vetergesic (0.05 mg kg À 1 , Alstoe Animal Health).The back skin was shaved and sterilized with Videne surgical scrub (Ecolab) then rinsed with sterile water.Then two full-thickness, 4 mm diameter back skin wounds with a minimum distance of 1 cm apart were made using a punch biopsy (Stiefel).Some wounds were topically treated daily with recombinant TNFa (8 mg kg À 1 , product code: PNRMTNFAI, Thermo Scientific Pierce), TNFa blocking antibody (10 mg kg À 1 , Adalimumab, Humira, Abbott Laboratories) or PBS until humanely sacrificed 2 or 5 dpw 49 .Some mice were injected intraperitoneally with anti-NRR1 (5 mg kg À 1 , Genentech), anti-NRR2 (5 mg kg À 1 , Genentech) or control (anti-ragweed, Genentech) IgG (5 mg kg À 1 ) for 7 days before wounding and then every 2-3 dpw for the duration of the experiment 30 .Some RORg À / À mice were injected intravenously with 1 Â 10 6 splenocytes derived from Rag2 À / À mice 24 h before wounding.Some Rag2 À / À mice were injected intraperitoneally with 200 ng anti-Thy1.2antibody (BioXcell, 30H12) every other day for 2 weeks before wounding 24,44 .Mice were kept up to 8 dpw and wounds were measured with a calliper or quantified using a 3D camera (Quantificare). Wounds were photographed using a 3D camera (Quantificare) during the healing response.Each wound was photographed three times at each time point, with each image analyzed three times to check for measurement consistency.Each image was assigned a random code and blind analysis was carried out using Dermapix software (Quantificare).Wound perimeters and diameters were recorded along with observations of the image (for example, excessive glare from the camera flash), and outliers excluded as appropriate when images could not be processed due to camera angle, excessive glare or obscured wound region. In every experiment mice were age and sex matched, with wildtype littermate controls used when possible, and within each experiment 3 or more mice per genotype/experimental group were included.Experiments were repeated two to four times to confirm results.The majority of mice reported were females between 6 and 9 weeks of age.Occasionally, male mice or mice between 9 and 18 weeks were included in an experiment, however RNA and protein analysis was performed on age and sex-matched tissues and analysis was performed among experimental replicates where appropriate.Unexpectedly, rare RORg À / À mice developed leukaemia while undergoing experimental procedures and these mice were excluded from analysis.In addition, some larger wounds were made by 6 mm punch biopsy (Supplementary Fig. 7f) for wound closure study. Antibody labelling.Tissue was collected and processed as previously described 9 .8 mm-thick frozen or paraffin sections were fixed in 4 or 0.4% paraformaldehyde, blocked in 10% goat or donkey serum, 0.25% fish skin gelatin and 0.2% bovine serum albumin, and then stained with antibodies diluted in blocking solution.Tissue sections were stained with fluorescent secondary antibodies (1:1,000, Invitrogen) and DAPI counterstained before imaging using a Leica Tandem SP5, Zeiss 710 or Zeiss 880 confocal microscope.For intracellular staining, frozen sections were fixed with 4% PFA, blocked as described above and incubated with fluorescence conjugated conjugated RORg antibody diluted in permeabilization buffer (eBiosciences, cat.00-8333) for 2 h.Brightness of images was adjusted using Adobe Photoshop CS3 software.See below for list of antibodies, suppliers and working dilutions. Flow cytometry.An 8 Â 8 mm piece of mouse back skin including wounded area was removed from and the ventral surface was scraped gently with a scalpel to remove subcutaneous fat and muscle.Tissue was chopped into pieces and incubated with 0.35 mg ml -1 Liberase TL (Roche, Cat: 5401020001), 3 mg ml À 1 Collagenase D (Roche, Cat: 11088866001) and 0.1 mg ml À 1 DNase I (Roche, Cat: 10104159001) in RPMI Media 1640 (Life Technologies, Cat: 31870-025) at 37 °C for 2 h.After incubation, the digested tissue was put through a 70 mm strainer to isolate single cells.Cells were pelleted and re-suspended in 0.5% BSA/2 mM EDTA in PBS, then were blocked with anti-mouse CD16/32 (Biolegend, Cat:101320) and rat IgG (Sigma, Cat: I8015-10 mg) for 20 min before antibody staining for flow cytometry.For intracellular staining of RORgt, cells were first stained for surface markers as described above, then fixed overnight in Foxp3 Fixation/Permeabilization (eBiosciences Cat: 88-8824).After incubation, a RORgt antibody (BD, Q31-378, 1:100) was added to the cells for 1 h before analyzing cells. For cytokine and chemokine staining, single cell suspensions were incubated with 50 ng ml À 1 PMA (Phorbol 12-myristate 13-acetate; Sigma, P1585) and 5 ng ml À 1 ionomycin (Sigma, 10634) in the presence of 10 mg ml À 1 brefeldin A (Sigma, B5936) for 3 h before surface staining as described above.Cells were fixed in Foxp3 Fixation/Permeablization buffer (eBioscience, 88-8824) overnight, then antibodies to IL22, IL17A, IL17F and/or MIP1a (CCL3) and isotype controls were added to the cells for 1 h before analyzing cells.Cells were analyzed using a BD LSR Fortessa.See below for list of antibodies, suppliers and working dilutions. Antibodies for mouse flow cytometry and immunohistochemistry. Flow cytometry.CD45 (Biolegend, 30-F11, 1:100), Lineage cocktail (eBioscience, inclu-ding17A2, RA3-6B2, M1/70, TER-119, RB6-8C5, 1:100), CD3 (eBioscience, 17A2, 1:100), CD3e (BD Biosciences, 145-2C11, 1:100), CD4 (eBioscience,RM4-5, 1:100), CD8 (Biolegend, 53-6.7,1:100), F4/80 (eBioscience, BM8,1:400), CD11b (Biolegend, M1/70, 1:1000), Gr-1 (eBiosiences, RB6-8C5, 1:100), NK1.1 (Biolegend, PK136), NKp46 (Biolegend, 29A1.4,1:100), CD117 (eBioscience, 2B8, 1:100), CD127 (eBioscience, A7R34, 1:100), CXCR5 (Biolegend, L138D7, 1:100), CXCR4 (Biolegend, L276F12, 1:100), CCR6 (Biolegend, 29-2L17, 1:100), ST2 (eBioscience, RMST2-2, 1:100), CD25 (Biolegend, PC61.5, 1:100), RORgt (BD, Q31-378, 1:100), IL22 (eBioscience, 1H8PWSR, 1:100), IL17A (Biolegend, TC11-18H10.1, 1:100), IL17F (Biolegend, 9D3.1C8, 1:100) and MIP1a (CCL3) (eBioscience, DNT3CC, 1:100). Immunohistochemistry. F4/80 (eBioscience, BM8,1:400), CD3 (eBioscience, 17A2, 1:100), CD4 (eBioscience, RM4-5, 1:100), CD117 (eBioscience, 2B8, 1:100), CD127 (eBioscience, A7R34, 1:100), RORgt (eBioscience, AFKJS-9, 1:100 or BD, Q31-378, 1:100), activated Notch1 (Abcam, ab8925, 1:200), Ki67 (Abcam or Novocastra, NCL-Ki67p, 1:400), Gr-1 (eBiosiences, clone BM8, 1:100), a6 integrin (Abcam, 105669, 1:400).Jagged 1 (Santa Cruz Biotechnology, SC-6011, 1:100) and K10 (Biolegend, 905401, 1:1000). Western blotting.A small region of the skin containing the wound and the region just adjacent to the wound (approximately 0.5-1 cm 2 ) was excised from the back skin.The tissue was homogenized using a polytron tissue homogenizer in RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 0.25% sodium deoxycholate with protease inhibitors).Lysate supernatants were run on a 6 or 10% polyacrylamide gel, transferred to PVDF membrane, blocked with 3% cold water fish skin gelatin (Sigma)/0.2%Tween-20/PBS and hybridized with antibodies to activated Notch1 (ab8925, 1:400, Abcam), activated Notch2 (ab8926, 1:400, Abcam) or beta-actin (A5441, 1:3000, Sigma).Blots were rinsed in 0.2% Tween-20/PBS, incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Sigma) and visualized with ECL Western Blotting Substrate (Pierce).Following film detection of blots, band intensity was quantified using ImageJ and the mean band intensity value was calculated against the loading control (Fig. 1 and Supplementary Fig. 5).Images have been cropped for presentation (Supplementary Fig. 5).Full-size images of western blots are presented in Supplementary Fig. 9. RNA extraction and quantitative polymerase chain reaction.Wounded back skin tissues were collected as above and RNA was extracted from freshly-isolated tissues, from tissues snap frozen in liquid nitrogen at the time of collection or from tissues immersed in RNAlater (Invitrogen, R0901) for 24 h before freezing.Tissues were homogenized using polytron tissue homogenizer then RNA was isolated using RNeasy Mini Kit (Qiagen, 74104) following manufacturer's instructions including the optional, on-column DNAse I digestion step using RNase-free DNase set (Qiagen, 79254).An additional Proteinase K digestion step was performed in the protocol: homogenized lysates were incubated with 0.2 mg ml À 1 Proteinase K (Sigma, P2308) in RLT buffer (Qiagen) at 55 °C for 10 min.Harvested RNAs were quantified using a NanoDrop microspectrophotometer and cDNA were prepared using High Capacity cDNA Transcription kit (Life Technologies, Cat: 4368814). qPCR was performed using a StepOnePlus Real-Time PCR System instrument (Life Technologies, Cat. 4376600), with a standard quantitation-comparative Ct procedure as set by the manufacturer.Triplicate reactions (25 ml) of each experimental sample were prepared using CYBR master mix (Life Technologies, Cat: 4367659) and unlabelled primers for CXCL12, CXCL13, CCL3 and HPRT (control).Reactions were subjected to an initial 10-minute denaturation step at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s.CCL20 and TNFa mRNA levels were examined using FAM probes and TaqMan Fast Master Mix (Life Technologies, 4352042) with a fast quantitation-comparative Ct procedure.Reference gene GAPDH was used as internal control.Reactions (20 ml) were subjected to an initial 20-s denaturation step at 95 °C, followed by 40 cycles of 95 °C for 3 s, 60 °C for 30 s. An alternative qPCR method to quantify CCL20 (Supplementary Fig. 6f) was performed using a Rotor-Gene Q instrument (Qiagen), with a two-step rapidcycling procedure as described by the manufacturer (Rotor-Gene Probe Handbook, Qiagen).Triplicate reactions (20 ml) of each experimental sample were analysed using a FAM probe for CCL20 and TaqMan Fast Universal PCR master mix (ABI).Reactions were subjected to an initial 3-minute denaturation step at 95 °C, followed by 45 cycles of 95 °C for 3 s and 60 °C for 10 s.Data were analysed using the Comparative Quantitation algorithm in the Rotor-Gene software, with calibrator samples for each run being compared in a common experiment.PCR reactions: 95 °C for 3 mins, followed by 45 cycles of 95 °C for 20s.A FAM probe for reference gene GAPDH was used to quantify genes of interest. Unlabelled primers and FAM probes for qPCR.All primers were used at a final concentration of 400 nM. CXCL12: Forward 5 0 -CAGAGCCAACGTCAAGCA-3 0 , Reverse 5 0 -AGGTA CTCTTGGATCCAC-3 0 CXCL13: Forward 5 0 -CATAGATCGGATTCAAGTTACGCC-3 0 , Reverse 5 0 -TCTTGGTCCAGATCACAACTTCA-3 0 CCL3: Forward 5 0 -GTTCTTCTCTGTACCATGAC-3 0 ; Reverse 5 0 -CTCTTAG TCAGGAAAATGAC-3 0 Control HPRT: Forward 5 0 -AGGAGTCCTGTTGATGTTGCCAG-3 0 , Reverse 5 0 -GGGACGCAGCAACTGACATTTCT-3 0 Jagged1 Forward: 5 0 -AGAAGTCAGAGTTCAGAGGCGTCC-3 0 , Reverse: 5 0 -AGTAGAAGGCTGTCACCAAGCAAC-3 0 Jagged2 Forward: 5 0 -CAATGACACCACTCCAGATGAG-3 0 , Reverse: 5 0 -GGC CAAAGAAGTCGTTGCG-3 0 FAM probes.IL22: Mm01226722_g1, Life Technologies CCL20: Mm01268754_m1, Life Technologies TNFa: Mm00443258_m1, Life Technologies GAPDH: Mm99999915_g1, Life Technologies Systems biology analysis of gene expression data sets.To determine key signalling pathways involved in Notch-mediated wound healing we used unbiased analysis of gene arrays from RORg þ innate lymphoid cells (NIH GEO: GSE29777) 31 and from uninjured, K14NICDER transgenic mouse back skin epidermis and dermis (NIH GEO: GSE23782) 9 .The MAS5-processed datasets of differentially expressed genes were imported into Ingenuity Pathway Analysis software (Ingenuity Systems, www.ingenuity.com) for analysis.All secreted, skin-derived factors that could interact with a receptor on ILC3s were determined. The highly upregulated genes in the epidermis and the dermis were filtered by the 'extracellular matrix' gene ontology term.Ingenuity Pathway Analysis was used to build direct connections from the upregulated genes to their possible target receptors.The genes displayed in Fig. 5 were further selected to show the most probable/relevant/interesting interactions between secreted factors and their target receptors. Human cutaneous wound biopsies.Full-thickness punch biopsies (diameter 5 mm) were taken from the medial aspect of the upper arm of five healthy ethically consented Caucasian volunteers (male and female; aged 18-28; REC ethics reference: 09/H1012/3).These were left to heal by secondary intention.After 3 and 7 days the initial wound sites were biopsied again (diameter 7 mm) so that the entire wound site was collected with some adjacent uninjured wound margin tissue.Samples were snap frozen in liquid nitrogen immediately following biopsy, embedded in OCT (CellPath, UK) and set on dry ice.Samples were stored at -80 °C until cryosectioning for further analysis.Cryosections of the tissue (6 mm) were allowed to reach room temperature and fixed in ice cold acetone at 4 °C for immunohistochemistry (IHC) or in formalin for H&E staining.Slides were then allowed to air dry at room temperature and washed in TBS before commencement of the immunohistochemical or histochemical protocol.For H&E, slides were first submerged in modified Harris Haematoxylin (Thermo Scientific, UK) for 4 min, washed in tap water and counterstained in Eosin (Thermo Scientific, UK) for 1 min.Stained slides were dehydrated through an ethanol gradient, cleared with xylene and mounted. For IHC, a sequential triple stain was performed.Slides were blocked in TBS with 10% (v/v) normal donkey serum and normal human serum.After three washes with TBS, a first incubation with mouse monoclonal Anti-RORC (1:50, MABF81, Millipore) and rabbit monoclonal anti-CD3 (1:100, AC-004 A, Epitomics) was performed for 1 h at room temperature.Sections were washed with TBS-tween-20 (0.01% (v/v) then incubated with secondary antibodies (Alexa-488 Donkey anti-mouse, 1:250; Alexa-555 Donkey anti-rabbit, 1:150) for 1 h at room temperature.After washing again, sections were blocked with TBS containing 10% (v/v) normal mouse serum.A second incubation with the third primary antibody, mouse monoclonal anti-CD127 (IL-7Ra) (1:100, A18684, Life Technologies), was then performed for 1 h at room temperature, followed by washing with TBS-Tween-20.Sections were then incubated with alexa-647 donkey anti-mouse (1:250) for 1 h at room temperature, washed and counterstained with DAPI (1:500) for 15 min at room temperature.Slides were given a final wash, dried and mounted using Vector Shield.Slides were scanned on a Pannoramic 250 Flash II, 3DHistech Ltd. and analysed through the Pannoramic Viewer platform (v.1.15.2 created by 3DHistech Ltd). Primary dermal cells.Primary mouse fibroblasts were isolated using a method on the basis of Jahoda et al. 65 .In brief, after sacrificing the mouse, the back skin hair was clipped and then the skin tissue and washed in DMEM (Invitrogen) with double strength antibiotics (2 Â Pen/Strep-Penicillin 100 U ml À 1 Streptomycin 100 mg ml À 1 ).The fat and non-dermal tissue was gently scraped from the tissue before cutting into pieces of B10 mm 2 .Each piece of tissue was cut repeatedly using curved blade scissors until it became slurry and the tissue was spread onto the bottom of a 6-well plate.The tissue was then covered with DMEM þ 10% foetal calf serum and 1 Â Pen/Strep (Fisher) and placed in a 37 °C incubator at 5% CO 2 .After 12 days cells were passaged. P1 mouse fibroblasts 90% confluent were treated with either 1:1,000 TNFa (20 ng ml À 1 , product code: PNRMTNFAI, Thermo Scientific Pierce), 1:1,000 TNFa blocking antibody (50 mg ml À 1 , Adalimumab, Humira, Abbott Laboratories) or both diluted in DMEM þ 10% FCS (PAA Laboratories) and 1 Â Pen/Strep.Some cells were left untreated.Cells were incubated for an additional 24 h then lysed and RNA collected using an RNAeasy Mini Kit (Qiagen) following the manufacturer's protocol. Statistics.For western blotting and mRNA quantification, samples were normalized where the average of the uninjured skin control, unless otherwise stated, was designated as 1 or 100%.The number of independent samples is stated in the figure legends.Statistical analysis was performed using SPSS (IBM) and Prism GraphPad software.Data variances were calculated and normal distribution of the data was determined by Kolmogorov-Smirnov tests.Normally-distributed data were analysed by two-way ANOVA with statistical significance between zones at calculated by Bonferroni's multiple comparison test or Student's t-test.Non-normally-distributed data was analysed using a Mann-Whitney test.% wound openness (Fig. 7b and Supplementary Fig. 7) was calculated using a linear mixed effects model.Specific P-values are included on figures or in figure legends. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/ How to cite this article: Li, Z. et al.Epidermal Notch1 recruits RORg þ group 3 innate lymphoid cells to orchestrate normal skin repair.Nat.Commun.7:11394 doi: 10.1038/ncomms11394 (2016).This work is licensed under a Creative Commons Attribution 4.0 International License.The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ ", "section_name": "Methods", "section_num": null } ]	[ { "section_content": "We wish to thank Paul Kaye, Jason Cyster, Kristin Braun, Fiona Watt and Clare Bennett for advice, reagents and manuscript feedback.Work was funded by the British Skin Foundation (2010s S.B., Z.L. and C.A.A.), BBSRC (BB/H02431X/1 I.C. and C.A.A.), Wellcome Trust (Computational Approaches in Translational Science WT095024MA G.L. and M.C.), HFSP (RGP0006/2009 T.C. and M.C.), MRC (MR/K021125/1 C.A.A, A.B. and M.C.C.; G0601156 M.C.C.). ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications Competing financial interests: The authors declare no competing financial interests. ", "section_name": "Author contributions", "section_num": null }, { "section_content": "", "section_name": "Author contributions", "section_num": null }, { "section_content": "Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications Competing financial interests: The authors declare no competing financial interests. ", "section_name": "Additional information", "section_num": null } ]
10.1007/s13205-022-03225-z	Diagnostic and prognostic potential clustered miRNAs in bladder cancer	<jats:title>Abstract</jats:title><jats:p>At specific genomic loci, miRNAs are in clusters and their association with copy number variations (CNVs) may exhibit abnormal expression in several cancers. Hence, the current study aims to understand the expression of miRNA clusters residing within CNVs and the regulation of their target genes in bladder cancer. To achieve this, we used extensive bioinformatics resources and performed an integrated analysis of recurrent CNVs, clustered miRNA expression, gene expression, and drug–gene interaction datasets. The study identified nine upregulated miRNA clusters that are residing on CNV gain regions and three miRNA clusters (hsa-mir-200c/mir-141, hsa-mir-216a/mir-217, and hsa-mir-15b/mir-16-2) are correlated with patient survival. These clustered miRNAs targeted 89 genes that were downregulated in bladder cancer. Moreover, network and gene enrichment analysis displayed 10 hub genes (CCND2, ETS1, FGF2, FN1, JAK2, JUN, KDR, NOTCH1, PTEN, and ZEB1) which have significant potential for diagnosis and prognosis of bladder cancer patients. Interestingly, hsa-mir-200c/mir-141 and hsa-mir-15b/mir-16-2 cluster candidates showed significant differences in their expression in stage-specific manner during cancer progression. Downregulation of NOTCH1 by hsa-mir-200c/mir-141 may also sensitize tumors to methotrexate thus suggesting potential chemotherapeutic options for bladder cancer subjects. To overcome some computational challenges and reduce the complexity in multistep big data analysis, we developed an automated pipeline called CmiRClustFinder v1.0 (<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://github.com/msls-bioinfo/CmiRClustFinder_v1.0">https://github.com/msls-bioinfo/CmiRClustFinder_v1.0</jats:ext-link>), which can perform integrated data analysis of 35 TCGA cancer types.</jats:p>	[ { "section_content": "Carcinoma in the epithelial lining of the urinary bladder has been increasing over the years (Sarkis et al. 2020).Globally, 573,278 bladder cancer (BCa) cases and associated 212,536 deaths were reported in 2020 (Sung et al. 2021).Traditional techniques for the diagnosis, prognosis, and monitoring of BCa include imaging tests (ultrasound, computerized tomography (CT), magnetic resonance imaging (MRI)), cystoscopy, and urine cytology.However, these methods fall short of expectations due to high cost, poor cytology sensitivity, high invasiveness of cystoscopy, and significant inter and intra-observer variability in tumor stage and grade interpretation (Su et al. 2019).The food and drug administration (FDA) has approved urinary biomarkers include BTA Stat, BTA Trak, nuclear matrix protein 22 (NMP22), UroVysion, ubiquitin C (UBC), and other assays such as immunocytochemistry (uCyt+ and DD23) and fluorescence in situ hybridization (FISH) being widely used for patient followup, though with lesser potency in low-grade cancer detection (Charpentier et al. 2021).BCa is generally managed with classical approaches such as chemotherapy, surgery, or radiation (Dobruch and Oszczudłowski 2021).Genetic and epigenetic alterations such as aberrant DNA methylation, altered chromatin remodeling, and dysregulated non-coding RNAs drive several molecular events during pathogenesis thereby contributing to varied clinical behavior in cancer recurrence and progression (Li et al. 2016;Martinez et al. 2019).Collectively, identifying novel biomarkers for prognosis and therapy is crucial to improving BCa patient care. MicroRNAs (miRNAs) are approximately 20-23nt long, highly conserved, and non-coding RNAs that play an important role in the regulation of gene expression.The miRBase (v22.1)currently consists of 2654 mature miRNAs coded from 1917 precursor miRNAs.Recent studies have shown that the human genome contains 159 miRNA clusters that comprise 468 miRNAs (Kozomara et al. 2019).A miRNA cluster consists of two or more miRNAs located in physically adjacent regions and transcribed in the same orientation.Differentially expressed clustered miRNAs have been reported.For example, cluster hsa-mir-143/mir-145 on chromosome location 5q32 is downregulated in several cancers (Das and Pillai 2015).Clinical and mechanistic studies have shown that the deregulated clustered miRNA expression may play a crucial role in the pathogenesis of BCa (Braicu et al. 2019).Therefore, differentially expressed clustered miRNAs can be used for diagnostic and prognostic purposes in BCa. Recent studies have reported that copy number variations (CNVs) are associated with aneuploidies and chromothripsis (Ben-David and Amon 2020) and are being recognized as an important risk factor for cancer, as they can alter the expression of their resident coding and non-coding genes (Shao et al. 2019).The miRNAs which reside on the CNV loci show a significant difference in expression patterns, especially in cancer conditions (Anauate et al. 2019).More than 50% of the miRNA genes are reported to overlap with the cancer-hotspot genomic regions and form a central regulatory unit in cancer development pathways (Farazi et al. 2013).The systematic array-based study by An et al. (2013) reported upregulation of two miRNA clusters (hsa-mir-23a/mir-24-2 and hsa-mir-181c/mir-181d) at chromosomal region 19p13.13due to CNV amplification in gastric cancer.Overexpression of the largest human miRNA cluster 'C19MC' has been linked to a variety of cancers, including breast cancer (Jinesh et al. 2018), brain tumors (Sin-Chan et al. 2019) and thyroid adenomas (Rippe et al. 2010).The co-localization of moderately explored CNVs and miRNAs has indicated the potential of CNV-mediated variation in C19MC miRNA dosage (Vaira et al. 2012). However, there are no comprehensive studies on CNV regulated clustered miRNA expression and its contribution to BCa.Hence, we performed an integrated analysis to identify potential clustered miRNAs to screen BCa.To meet the objectives, we performed an integrated analysis using a) clustered miRNAs residing on CNVs, b) patient survival, c) miRNA targeted genes, d) gene function and e) drug-gene interaction.We have developed a user-friendly computational pipeline named 'CmiRClustFinder v1.0' by integrating R and shell scripts.The pipeline can be effectively used for high throughput data analytics and to identify biomarkers for cancer diagnosis. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Materials and methods", "section_num": null }, { "section_content": "Level 3 miRNA expression datasets of BCa were interrogated from the TCGA database (https:// portal.gdc.cancer.gov/ proje cts/ TCGA-BLCA).We analyzed 412 BCa patients data, of which 304 are males and 108 are females.The data belong to White (327 samples), Asian (44), Black or African-American (23) populations, and 18 samples population information was not available.Pre-computed Somatic copy-number alterations (SCNA) data without germline copy number amplification (CNA) was obtained from the Broad Institute's FireBrowse portal (http:// fireb rowse.org/).Further, 468 miRNAs belonging to 159 clusters were retrieved from the miRBase V22.1 (http:// www.mirba se.org/) database. ", "section_name": "Acquisition of miRNA expression and CNV data from bladder carcinoma", "section_num": null }, { "section_content": "Recurrent SCNAs in the TCGA-BLCA samples were analyzed using Bioconductor package GAIA 3.10 (https:// bioco nduct or.org/ packa ges/ relea se/ bioc/ html/ gaia.html).Recurrent CNV was defined by false discovery rate (FDR) Q < 0.15 derived from 10 iterations.The segment mean of 0.3 was set as the threshold to identify the copy number gain/loss.The regions with segment mean > 0.3 and ≤ 0.3 thresholds were considered as a copy number gain (amplification) and loss (deletion) respectively.Finally, the genomic SCNA plot was generated using an R script with the cut-off value < 0.15 FDR.The GAIA uses human genome assembly Hg19 for all the analyses.Hence, we used the UCSC LiftOver (https:// genome.ucsc.edu/ cgi-bin/ hgLif tOver) option to lift all SCNA genomic coordinates to match with the Hg38 build.Further, the functionality of BEDTools (Quinlan and Hall 2010) was used to intersect the genomic coordinates of miRNA clusters onto the recurrent significant CNV regions identified from GAIA analysis. ", "section_name": "Identification of SCNA and miRNA cluster co-occurrence", "section_num": null }, { "section_content": "We used various functions of the 'TCGAanalyze_DEA' and TCGAbiolinks packages (Robinson et al. 2010) to identify differentially expressed miRNAs (Normal vs. Tumor).Further, using the false discovery rate (FDR) correction, the p value was adjusted to shortlist the top differentially expressed miRNAs.The logFC > 1 and FDR < 0.05 threshold were considered to be significant. ", "section_name": "The miRNA differential expression analysis", "section_num": null }, { "section_content": "Following criteria were used to identify the differentially expressed miRNA clusters residing on CNV regions: (i) at least two miRNAs must be differentially expressed from a cluster, and (ii) differentially expressed miRNA clusters must reside on copy number amplified or deleted regions.Inversely correlated cluster expression with CNVs and clusters with non-significant expression were excluded from the further analysis. ", "section_name": "Identification of clustered miRNA expression residing on CNV", "section_num": null }, { "section_content": "The BCa samples were classified into low and high expression groups to identify the prognostically significant miRNA clusters according to the median miRNA expression levels.The Kaplan-Meier plotter hosted by the miRpower tool (Lánczky et al. 2016) was used to determine the relapsefree survival (RFS) with different clinical parameters.For miRNA cluster expression analysis, an unpaired t test was performed using MedCalc version 15.0 (Schoonjans et al. 1995).The p value < 0.05 was considered statistically significant.The plotted curves such as receiving operator characteristic (ROC) and areas under ROC (AUROC) were evaluated by comparing the values from tumor and normal tissues. ", "section_name": "Identification of prognostic signatures", "section_num": null }, { "section_content": "The target genes of survival correlated miRNAs were obtained from miRTarBase (Huang et al. 2020), TargetScan (Garcia et al. 2011), DIANA-TarBase (Vlachos et al. 2015), mirDIP (Tokar et al. 2018), and miRDB (Chen and Wang 2020).Target genes commonly found in these five databases were only considered for further downstream analysis.The target gene expression and gene promoter methylation information were mined from the UALCAN database (http:// ualcan.path.uab.edu/).Networks of downregulated genes targeted by clustered miRNAs were plotted.Further, these genes were examined for DNA promoter methylation to confirm the miRNAs target effect on gene expression.Using the beta value given in the UALCAN web server, DNA methylation was estimated in a range of 0 (unmethylated) to 1 (methylated). ", "section_name": "miRNA target prediction and regulatory network analysis", "section_num": null }, { "section_content": "The clustered miRNAs and their target genes network interaction were analyzed with GeneMANIA (https:// genem ania.org/) to explore the co-expression, co-localization, and shared protein domain information.The PPI networks were constructed using STRING V11.0 (Szklarczyk et al. 2017) with a medium confidence score ≥ 0.40 to predict the most interactive genes.The PPI network was imported and visualized using the Cytoscape plugin Strin-gApp (Doncheva et al. 2018).Using CytoHubba (Chin et al. 2014), the top 10 hub genes were identified based on the distribution of network node degrees. ", "section_name": "Protein-protein interaction (PPI) network analysis", "section_num": null }, { "section_content": "We performed the functional enrichment analysis for 89 target genes using DAVID (https:// david.ncifc rf.gov/) platform.The p value calculated using the Benjamini-Hochberg method and ≤ 0.05 threshold was considered statistically significant. ", "section_name": "Gene set enrichment analysis", "section_num": null }, { "section_content": "The present study used bladder cancer (404 samples) and normal tissue (28 samples) datasets available at the GEPIA server (Tang et al. 2017).The hub genes differentially expressed with a p value < 0.01 were considered statistically significant.The boxplot was used to illustrate the association between cancer and normal tissues.We used 412 BCa patient data available at the TCGA-GDC portal for the molecular subtype-specific expression analysis.These datasets comprised into five subtypes, basalsquamous (n = 142), luminal-papillary (n = 142), luminalinfiltrated (n = 78), luminal (n = 26), neuronal (n = 20) and four samples were uncategorized.To gain better insights, we have studied the BCa subtype-specific expression profile for three miRNA clusters and 10 hub genes.Tobacco smoking is a high-risk factor for carcinogenesis, and it can affect various organs such as the head, neck, lungs, and urinary bladder.Also, smoking induces the expression of various miRNAs, which post-transcriptionally silence the function of tumor suppressors and promote cancer (Fujii et al. 2018).Altered miRNA expression contributes to tumor growth and plays a critical role in response to chemotherapy (Li et al. 2013).Hence, we have studied the clustered miRNA and hub gene expression profiles in patients categorized into (i) smokers and non-smokers, (ii) chemotherapy responders, and non-responders. ", "section_name": "Tissue, molecular-subtype, and clinical traits specific expression analysis", "section_num": null }, { "section_content": "To validate the expression of clustered miRNAs and their target genes, we have used one miRNA-GSE36121 (Ratert et al. 2012); and three mRNA datasets-GSE40355 (Hecker et al. 2013), GSE27448 (Lambrou et al. 2013), GSE52519 (Borisov et al. 2018).These datasets were considered as the comprehensive reference for the miRNA and gene expression studies in urothelial carcinomas (Normal vs Tumor).The R package Limma-based online program GEO2R (https:// www.ncbi.nlm.nih.gov/ geo/ geo2r/) was used to perform differential expression.The p value < 0.05 and logFC > 1 considered as significant. ", "section_name": "In silico validation of miRNA and gene expression", "section_num": null }, { "section_content": "The hub genes were analyzed with the DGIdb database (http:// www.dgidb.org/) to understand the interrelation between drug candidates and genes.The analysis was restricted to the drugs approved by the Food and Drug Administration (FDA).Further, the PanDrugs (https:// www.pandr ugs.org/) database was used to identify potentially druggable molecular alterations and prioritization of anticancer drugs. ", "section_name": "Hub-genes and drug interaction", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "The GAIA analysis of BCa-SCNA data resulted in 4119 significant CNV aberrations, of which 1824 regions were amplified, and 2295 regions were deleted (Supplementary Table S1, Fig. 1A).The mapping of 159 miRNA clusters on CNV regions showed overlapping of 61 clusters with CNV regions (Supplementary Table S2).Of the 61 miRNA clusters residing on CNV regions, 33 and 28 were located on amplified and deleted regions, respectively.Further, the expression analysis of quantile normalized BCa vs normal samples displayed 661 upregulated and 33 downregulated miRNAs.Using an integrated analysis, we identified 41 miRNAs belongs to nine upregulated miRNA clusters (hsa-mir-7113/mir-4691, hsa-mir-200c/ mir-141, hsa-mir-3913-1/mir-3913-2, hsa-mir-657/mir-1250, hsa-mir-512-1/mir-526a-1, hsa-mir-371a/mir-373, hsa-mir-6804/mir-6803, hsa-mir-217/mir-216a and hsamir-15b/mir-16-2) which reside on the CNV gain regions (Supplementary Tables S3 &S4).However, no statistically significant expression was observed for miRNA residing on CNV deleted regions. ", "section_name": "Identification of CNV driven miRNA clusters", "section_num": null }, { "section_content": "The Kaplan-Meier survival analysis was performed to identify the impact of CNV driven miRNA clusters on survival rate of BCa patients.We have compared survival analysis and hazard ratio with populations designated as miRNA clusters high and low risk in the TCGA database.The combined KM and ROC curve analysis identified three prognostically important miRNA clusters hsa-mir-200c/mir-141, hsa-mir-216a/mir-217, and hsa-mir-15b/mir-16-2 (Fig. 1B).The three miRNA clusters consist of six miRNAs were found to be upregulated (Supplementary Table S4).The overall survival rate of candidates of the hsa-mir-200c/mir-141 cluster is-hsa-mir-200c: HR = 0.57, CI = 0.42-0.78,p = 0.00036; hsa-mir-141: HR = 0.62, CI = 0.46-0.084,p = 0.0019.The candidate miRNAs of cluster hsa-mir-216a/mir-217 is-hsamir-216a: HR = 1.76,CI = 1.3-2.37,p = 2e-04; hsa-mir-217: HR = 1.39,CI = 1.03-1.88,p = 0.032 and for cluster hsa-mir-15b/mir-16-2 is-hsa-mir-15b: HR = 0.88, CI = 0.65-1.19,p = 0.42; hsa-mir-16-2: HR = 0.81, CI = 0.6-1.1,p = 0.18.The hsa-mir-200c/mir-141 and hsa-mir-216a/mir-217 (hsamir-200c: 2.01 LogFC; hsa-miR-141: 3.09 LogFC; hsa-mir-216a: 2.69 LogFC; hsa-mir-217: 2.45 LogFC) showed a significant correlation in the BCa patient survival (Fig. 2A,B).On the other hand, the hsa-mir-15b/mir-16-2 (hsa-mir-15b: 1.49 LogFC, hsa-mir-16-2: 1.52 LogFC) was not statistically significant for the survival of BCa patients (Fig. 2C).The higher expression of the hsa-mir-200c/mir-141 correlated with a better survival rate.However, hsa-mir-216a/ mir-217 overexpression lead to a significant reduction in patient's survival.We evaluated diagnostic value of CNV clusters by ROC curve analysis.The results indicated that the candidates of miRNA clusters hsa-mir-200c/mir-141 and hsa-mir-15b/mir-16-2 exhibited high diagnostic value (Fig. 2D,E).The area under the ROC curve (AUROC) for candidate miRNAs of these clusters were 0.784 (hsa-mir-200c), 0.894 (hsa-mir-141), 0.726 (hsa-mir-15b) and 0.752 (hsa-mir-16-2).The hsa-mir-216a/mir-217 cluster candidates harbored lower specificity and sensitivity (Fig. 2F) than the other two clusters.The conclusive KM and ROC results indicated that the prognostic model of members of these three clusters was robust in predicting the progression of cancer cell growth in BCa patients. ", "section_name": "Identification of the prognostic signature", "section_num": null }, { "section_content": "The CNV driven, overexpressed three miRNA clusters target 180 genes.Promoter methylation (beta value) levels and the expression profile for all the target genes were given in Supplementary Tables S5 & S6.Among these, 64 genes were upregulated, and 27 were hyper-methylated.Hence, these 91 target genes were excluded from further analysis. The 89 genes downregulated in BCa targeted by clustered miRNAs from CNV gain regions are illustrated in Fig. 3.We assume that these genes were downregulated due to the upregulation of miRNA clusters.The expression of 89 target genes was cross-checked with BCa patient data available in GEO cohorts.Interestingly, 74 genes were significantly downregulated in BCa datasets (Supplementary Table S6).However, expression data for 15 genes were not found in the selected GEO datasets. ", "section_name": "Prediction of miRNA target genes and construction of regulatory networks", "section_num": null }, { "section_content": "We used the strength of existing network-based tools to study the protein-protein interaction and biological significance.The GeneMANIA resulted in genes and protein network shown in Fig. 4A highlights the functional association.Functionally associated interactions of 89 genes targeted by three miRNA clusters are given in Supplementary Table S7.According to STRING results, the PPI network of hsa-mir-200c/mir-141 cluster targets consists of 31 nodes and 36 edges, hsa-mir-216a/mir-217 cluster targets comprised 20 nodes and nine edges, and cluster hsa-mir-15b/mir-16-2 consists of 40 nodes and 23 edges.The connectivity degree of each node was calculated in these PPI networks and node degree > 10 was considered for hub genes (CCND2, ETS1, FGF2, FN1, JAK2, JUN, KDR, NOTCH1, PTEN, and ZEB1).All the hub genes showed a strong association with their node proteins (Fig. 4B).However, among these hub genes, PTEN showed the highest node degree (20), which is targeted by both the candidates of cluster hsa-mir-216a/ mir-217.Among these hub genes, PTEN and ZEB1 are targeted by both the members of hsa-mir-216a/mir-217 and hsa-mir-200c/mir-141 clusters, respectively.Interestingly, mir-200c alone targets five of these hub genes (ETS1, FN1, JUN, KDR, and NOTCH1). ", "section_name": "PPI network construction and identification of hub genes", "section_num": null }, { "section_content": "Gene ontology enrichment analysis of 89 genes targeted by three miRNA clusters is given in Supplementary Table S8.These genes are associated with 64 biological processes (BP), notably positive and negative regulation of cell proliferation, cell migration, signaling pathways, and positive regulation of gene expression.The enriched molecular functions (MF) include DNA binding, RNA binding, protein kinase activity, growth factor activity, metal ion binding, zinc ion binding, and translation repression.The enriched cellular component (CC) includes cytosol, cytoplasm, nucleoplasm, nucleus, centrosomes, membrane-bound vesicles, and cell projection.Furthermore, these genes were significantly associated with 14 pathways, including cancer signaling pathways such as p53, PI3K-Akt, MAPK, Wnt, and ", "section_name": "GO term enrichment and KEGG pathway analysis", "section_num": null }, { "section_content": "Alongside this, the present study estimated the tissue-specific expression of hub genes in normal and bladder cancer tissues.The analysis also highlighted that ZEB1, CCND2, FGF2, JAK2, and JUN hub genes were significantly downregulated in BCa tissues (Fig. 6).Further, the examination of the CNV status of 10 hub genes showed that five genes reside on CNV regions (amplified: CCND2, KDR, and ZEB1; deleted: FN1 and PTEN).Hence, we hypothesize that the combined effect of CNV loss and miRNA binding might be responsible for the downregulation of FN1 and PTEN in BCa tissues.Subtype-specific expression patterns of clustered miRNA and hub genes were analyzed in BCa patients (Supplementary Fig. 1,2).Proportionally, the total number of patients having downregulation (subtype-specific) of hub genes is more except FN1 (Basal Squamous & Luminal Infiltrated), KDR (Luminal, Luminal Infiltrated & Luminal Papillary), NOTCH1 (Basal Squamous).Expression profile analysis based on the smoking history of BCa patients vs. normal samples showed significant upregulation of mir-200c/141 and mir-15b/16-2 clusters in all the categories.We have observed expression trends of cluster targeted 10 hub genes are significantly downregulated in all categories of smoking history (Supplementary Fig. 3A).The BCa patient's chemotherapy response trait information was procured for 236 samples from TCGA-GDC.Three miRNA clusters were upregulated in the responders and non-responders category except a miRNA mir-217 from mir-216a/217 cluster, which did not show a significant difference in expression compared with normal (Supplementary Fig. 3B).Interestingly, in the partial response group, NOTCH1 showed significant downregulation, but in the progressive and stable disease category, the expression was higher.Except for FN1, all other hub genes were downregulated in the therapy response category. ", "section_name": "Tissue, molecular-subtype, and clinical traits specific expression analysis", "section_num": null }, { "section_content": "The integrated analysis of drug-gene interactions showed an association of seven hub genes with 20 potential FDAapproved drugs (Supplementary Table S9).Further, hub genes were analyzed for prioritizing the drugs and their clinical actionability for cancer therapy.Using this analysis, a total of 242 experimental drugs, 25 clinical trials, and one FDA-approved potential drug (methotrexate) were interacting with hub genes (Supplementary Table S10).These results could provide an opportunity for the repurposing of the drugs to treat BCa. ", "section_name": "Hub-genes and drug interactions", "section_num": null }, { "section_content": "We have explored the expression pattern of CNV driven miRNA clusters in different stages of BCa (Supplementary Fig. 4).The stage-wise expression profile of both the candidates of hsa-mir-200c/mir-141 showed distinct signatures among BCa stages when compared to normal tissues (Supplementary Fig. 4A).The hsa-mir-15b/mir-16-2 candidates showed differences in the expression patterns in each stage of cancer progression (Supplementary Fig. 4C).Our analysis indicated that the candidate miRNA expression signature of hsa-mir-200c/mir-141 and hsa-mir-15b/ mir-16-2 could differentiate healthy tissues from malignant phenotypes. ", "section_name": "Utility of the miRNA clusters for BCa staging", "section_num": null }, { "section_content": "Big data analytics through command line computation may be a daunting task to life science researchers.Hence, to overcome the computational challenges and reduce the complexity of multistep Commandline computing, we developed an automated pipeline called CmiRClustFinder v1.0 that computes the integrated data within five steps (Fig. 7).The installation script will download all the necessary resources and prepare the pipeline for use in the first step.In the second step, the GAIA package finds frequent aberrations in chromosomal regions among cancer patients' datasets.In the third step, the LiftOver tool matches the genomic build for RCNVs and user-defined genetic elements.We have integrated BEDTools to find co-localization of significant RCNV and genomic elements in the fourth step.Lastly, the Circalize package generates a circos representation of the data.The source code for CmiRClustFinder v1.0 is publicly available at https:// github.com/ msls-bioin fo/ CmiRC lustF inder_ v1.0.The manual for the pipeline execution is available in the portal. ", "section_name": "CmiRClustFinder v1.0", "section_num": null }, { "section_content": "Abnormal changes in miRNA expression may play a crucial role in the initiation, development, and progression of various cancers (Farazi et al. 2013).Since miRNA clusters Fig. 7 Five steps working process of the CmiRClustFinder v1.0 automated pipeline contain multiple miRNAs encoding genes, their abnormal expression may collectively show a more severe impact on the cell signaling pathways than the individual miRNA.Recent reports have demonstrated clustered miRNAs cooperative and synergistic activity in various cancers (Cantini et al. 2019;Rui et al. 2020).Therefore, it is essential to explore the biological processes and cell signaling pathways affected due to dysregulated clustered miRNAs.Anauate et al. (2019) demonstrated copy number gain associated miRNA cluster of four miRNAs (miR-1207-3p, miR-1205, miR-1207-5p, and miR-1208) at 8q24 were upregulated ~ 50% of gastric tumors (Anauate et al. 2019).Our analysis identified 61 miRNA clusters that are localized within the CNV regions in BCa.To the best of our knowledge, this is the first comprehensive in silico study investigating the effect of recurrent CNV aberrations on miRNA cluster expression in BCa. We identified prognostically significant three miRNA clusters, hsa-mir-200c/mir-141, hsa-mir-216a/mir-217, and hsa-mir-15b/mir-16-2 from BCa patients.Each cluster comprises of two miRNAs localized within the recurrent CNV gain regions were overexpressed.The aberrant expression of hsa-mir-200c/mir-141 has been reported in many human malignancies, where it participates in a variety of cellular processes such as epithelial-mesenchymal transition (EMT), proliferation, migration, invasion, and drug resistance (Senfter et al. 2016).Additionally, this cluster has been established as a potential diagnostic and prognostic biomarker for various carcinomas such as ovarian cancer (Gao et al. 2015), breast cancer (Choi et al. 2016), and lung adenocarcinoma (Tejero et al. 2014).Although the prognostic impact of hsa-mir-216a/mir-217 and hsa-mir-15b/mir-16-2 candidates' dysregulation has been confirmed in several types of cancers (Aqeilan et al. 2010;Lovat et al. 2015;Azevedo-Pouly et al. 2017;Erener et al. 2021).However, there is no explicit evidence for these clusters can be used as a marker for BCa diagnosis. Here, log-rank test-based Kaplan-Meier survival analysis showed hsa-mir-200c/mir-141 and hsa-mir-216a/mir-217 to be significantly correlated with the overall survival rate of BCa patients.The overexpression of mir-200c and mir-141 is strongly associated with a better prognosis in bladder cancer patients.Our findings are consistent with Mei et al. 2020, with additional evidence for CNV-induced overexpression of hsa-mir-200c/mir-141. ROC/AUROC analysis indicated high diagnostic accuracy for hsa-mir-200c/mir-141 and hsa-mir-15b/mir-16-2 cluster members.On the other hand, members of the hsa-mir-216a/mir-217 cluster exhibited no substantial sensitivity or specificity. Further, members of these three miRNA clusters were subjected to target prediction, miRNA-mRNA network construction, PPI network analysis, pathway enrichment, and molecular function analysis to gain more insights.The study plotted a functional enrichment analysis of 89 genes that were targeted by three miRNA clusters to illustrate their association with different cancer signaling pathways.Network genes were filtered based on the number of nodes, and the top 10 higher degree node genes were denoted as hub genes (CCND2, ETS1, FGF2, FN1, JAK2, JUN, KDR, NOTCH1, PTEN, and ZEB1).The GO term and KEGG pathway enrichment analysis results indicated that these hub genes were related to the classical cancer signaling pathways.This includes previously defined p53, Ras, PI3K-Akt, and prolactin signaling.These pathways are closely correlated with proliferation, migration, invasion, and differentiation of the cancer cells (Sever and Brugge et al. 2015). Following tissue-specific gene expression analysis from the GEPIA, GEO, and ULCAN datasets, the hub genes were shown to be considerably downregulated.The DNA promoter methylation analysis showed that these hub genes are not hyper-methylated in BCa.We observed that CCND2, KDR, and ZEB1 genes are residing on the CNV gain region while FN1 and PTEN are on CNV loss.The combined effect of CNV loss and miRNAs binding might be downregulating FN1 and PTEN in BCa tissues.The target identification studies showed that 109 non-clustered miRNAs are also targeting these 10 hub genes.However, the expression of these 109 non-clustered miRNAs was not statistically significant or less expressed in BCa tissues when compared to the six clustered miRNAs (Supplementary Table S11).Hence, the integrated analysis hypothesizes that miRNA clusters residing on CNV gain regions are potential regulators for bladder cancer. Tobacco smoking is one of the most important risk factors for BCa with an attributable risk of approximately 50% (Cumberbatch et al. 2016).Tobacco-rich compounds such as aromatic amines and N-nitroso can induce DNA damage in the form of double-strand breaks, base modifications, or bulky adduct formation (Stern et al. 2009).Recent studies have suggested the possibility of tobacco smoking-induced oncogenic or antioncogenic gene expression by miRNA regulation in BCa (Cumberbatch et al. 2018;Navratilova et al. 2020).Hence, the analysis of the effect of recurrent genetic aberration in smoking and non-smoking groups of patients is essential.Considering this, BCa patients were grouped into five smoking categories (Lifelong Non-Smoker; Current Smoker; Current Reformed Smoker > 15 Years; Current Reformed Smoker ≤ 15 Years and Current Reformed Smoker; duration not specified) and expression analysis was performed.The members of mir-200c/mir-141 and mir-15b/ mir-16-2 showed significant upregulation in each category.In comparison, no significant category-wise difference in mir-216a/mir-217 expression was identified.With the exception of FN1, the expression of other hub genes was significantly downregulated in all smoking groups. The upregulated hsa-mir-200c/mir-141 targeted six hub genes: ETS1, FN1, JUN, KDR, NOTCH1, and ZEB1; all of which were downregulated in BCa.However, cancer stagewise analysis showed decreased expression for hsa-mir-200c/ mir-141 from stages 1 to 4 (Supplementary Fig. 4).In BCa, cluster hsa-mir-200c/mir-141 is frequently overexpressed and associated with early-stage (T1) bladder tumors (Han et al. 2011). Martínez-Fernández et al. (2015), suggested that increased expression of polycomb group protein BMI1 and EZH2 may contribute to the downregulation of hsamir-200c/mir-141 in high-grade stage BCa tumors through transcriptional repression.Downregulation of hsa-mir-200c/ mir-141 leads to a subsequent upregulation of EMT promoting transcription factors (ZEB1 and ZEB2) and thus favors the invasive behavior of the tumor cells and cancer progression (Martínez-Fernández et al. 2015).Overexpression of hsa-mir-200c/mir-141 showed an improved survival rate in BCa patients with high specificity and sensitivity, suggesting it as a potential marker for BCa diagnosis. The hsa-mir-15b/mir-16-2 cluster targeted CCND2 and FGF2 genes, were downregulated in BCa.The hyperactivation of CCND2 is generally considered as increased oncogenic activity in various tumors (Takano et al. 1999(Takano et al. , 2000)).Methylation of promoter region mediated silencing of CCND2 expression associated with a few cancer types progression is also reported (Evron et al. 2001;Wang et al. 2016).In the current analysis, identified CCND2 is unmethylated and resides on the CNV gain region in the tested bladder carcinoma datasets.Hence, we strongly assume that the downregulation of CCND2 might be due to the aberrant overexpression of hsa-mir-15b/mir-16-2 in malignant tumors. The detailed functional annotation revealed that hsa-mir-216a/mir-217 targeted two hub genes (PTEN and JAK2), which act as tumor suppressors in numerous cancers (Qian et al. 2011;Lu et al. 2016).Studies have been reported that miRNAs can perform an oncogenic role by suppressing the function of PTEN in BCa (Feng et al. 2014), and the PTEN gene found to be associated with the CNV loss region.Therefore, the actual mechanism behind the downregulation of PTEN genes needs to be further investigated.Collectively, these findings strongly suggest that CNV driven overexpressed clustered miRNAs play an important role in regulating BCa signaling genes. Resistance to treatment is one of the key problems associated with BCa patient survival.According to recent reports, miRNAs can also play a crucial role in the chemoresistance mechanism in BCa (Senfter et al. 2016;Cai et al. 2019).Using drug-gene interaction analysis, we have identified 20 potential FDA-approved drugs interacting with miRNA clusters and their targeted genes in BCa.The PanDrugs drug-gene interactions analysis suggests that molecular alterations in the NOTCH1 gene are associated with high sensitivity to methotrexate (MTX).The MTX is commonly used as an anti-metabolite and chemotherapy for several cancer types (Hagner and Joerger 2020).High expression of Notch signaling genes has a vital role in many tumor cell's resistance to methotrexate, while its downregulation increases drug sensitivity (Ma et al. 2013;Zhao et al. 2020).In the analysis of therapy responder and non-responder patients, we observed an upregulated expression of NOTCH1 in progressive and stable disease groups.Interestingly, in the disease progression group, the mir-200c/mir-141 was slightly downregulated.It suggests that the expression of mir-200c/mir-141 and NOTCH1 is inversely correlated.Also, in the partially responded group, NOTCH1 expression was downregulated.Hence, we assume that has-mir-200c/mir-141 cluster has a regulatory impact on NOTCH1, which may sensitize BCa cells to methotrexate. In this in silico study, we have demonstrated the relationship between recurrent CNVs gain and their effect on the upregulation of miRNA clusters in BCa.The BCa stage-wise expression pattern of miRNA cluster candidates of has-mir-200c/mir-141 and hsa-mir-15b/mir-16-2 showed a distinct signature to differentiate healthy individuals from malignant phenotypes.These miRNA signatures can be used as potential prognostic BCa markers and cancer treatment.Collectively, the miRNA clusters upregulated by CNV gain may downregulate several cancer signaling genes and sensitize cancer cells to methotrexate. Here, we report the first version of CmiRClustFinder, a computational pipeline that integrates multi-omics datasets such as CNV, miRNA, and gene expression to infer CNV driven clustered miRNAs from the TCGA cancer types.The CmiRClustFinder can be effectively utilized to identify novel miRNA biomarkers for various cancer types. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Multi-omics approaches provided a large volume of genetic data for life science knowledge discovery.The study analyzed clustered miRNAs residing on CNV regions and developed an automated pipeline for the integrated data analysis.The integrated CNV-miRNA clusters data analysis identified 61 miRNA clusters (consisting of 153 miRNAs) residing on the CNV gain/loss region.The CNV driven, three prognostically significant miRNA clusters (hsa-mir-200c/141, hsa-mir-216a/mir-217, and hsa-mir-15b/mir-16-2) showed 2-fivefold increased expression in bladder cancer.Further, hsa-mir-200c/mir-141 and hsa-mir-15b/mir-16-2 clusters showed stage-wise difference in cancer progression.Interestingly, these clustered miRNAs targeted top 10 hub genes, the expression of which was downregulated in BCa tissues.Functional annotation indicates these hub genes have a key role in BCa and significantly impact patient survival and diagnosis.Hence, we hypothesize that these dysregulated clustered miRNAs can be used to screen bladder cancer progression as a potential diagnostic and prognostic indicator.Moreover, our integrated in silico results highlight a potential therapeutic application of clustered miRNA-based therapies for bladder cancer.The integrated analysis observed silencing of NOTCH1 by mir-200c/mir-141 improves methotrexate treatment and could benefit the BCa patient's survival.The CmiRClustFinder pipeline can be used to identify novel clustered miRNAs to diagnose various cancer types. ", "section_name": "Conclusion", "section_num": null } ]	[ { "section_content": "The authors would like to thank DST-FIST, the Government of India, TIFAC-CORE in Pharmacogenomics, and Manipal Academy of Higher Education (MAHE), Manipal, for the support and facilities provided.Mr. Akshay acknowledges MAHE for the Dr. TMA Pai Ph.D. fellowship. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Funding Open access funding provided by Manipal Academy of Higher Education, Manipal.This work was supported by the Vision Group on Science and Technology (VGST), Government of Karnataka (KSTePS/VGST/2020-21/RGS/F/GRD-997/94/2021-22/944). ", "section_name": "", "section_num": "" }, { "section_content": "Code for data analysis used in this study is provided on GitHub in the form of a pipeline.https:// github.com/ msls-bioin fo/ CmiRC lustF inder_ v1.0. ", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1007/ s13205-022-03225-z. Author contributions KS designed the study; BP, SPK, AC coordinated the project; APW performed the data analysis and wrote the manuscript; BP, SPK, KS proofread and edited the manuscript; All authors read and approved the final version of the manuscript. Conflict of interest The authors declare no conflict of interest, financial or otherwise. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http:// creat iveco mmons.org/ licen ses/ by/4.0/. ", "section_name": "Supplementary Information", "section_num": null }, { "section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1007/ s13205-022-03225-z. Author contributions KS designed the study; BP, SPK, AC coordinated the project; APW performed the data analysis and wrote the manuscript; BP, SPK, KS proofread and edited the manuscript; All authors read and approved the final version of the manuscript. ", "section_name": "Supplementary Information", "section_num": null }, { "section_content": "Conflict of interest The authors declare no conflict of interest, financial or otherwise. ", "section_name": "Declarations", "section_num": null }, { "section_content": "Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http:// creat iveco mmons.org/ licen ses/ by/4.0/. ", "section_name": "Ethical approval The manuscript does not involve any animal study.", "section_num": null } ]
10.3960/jslrt.23032	Treatment patterns in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma post covalent Bruton tyrosine kinase inhibitor treatment: a Japanese claims database study	Standard treatment has not been established for patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after discontinuation of covalent Bruton tyrosine kinase inhibitor (cBTKi) therapy. This retrospective, administrative database (Medical Data Vision) study described the patient characteristics, treatment patterns, and factors associated with receiving post-first-cBTKi treatment in Japanese patients with CLL/SLL. Patients aged ≥18 years with confirmed CLL/SLL diagnosis and treated with anti-neoplastic drugs indicated for CLL/SLL between March 2013 and February 2022 were included. Patient characteristics at baseline (first line), first cBTKi exposure (first-cBTKi), post-first-cBTKi treatment received, and the treatment sequence of CLL drugs received first line through third line, were described. Time-to-event analyses used the Kaplan-Meier method. Multivariable logistic regression analysis was used to explore factors associated with receiving post-first-cBTKi treatment among patients who discontinued first-cBTKi treatment. Among 2,424 eligible patients (median age: 72.0 years, 61.9% male), 450 (18.6%) received cBTKi in any treatment line. Among patients treated with cBTKi, 273 (60.7%) discontinued treatment; 56.0% of them (n = 153/273) received subsequent treatment. Median duration of post-first-cBTKi treatment was 2.2 months (95% confidence interval [CI]: 1.8, 3.5). The most common regimens post-first-cBTKi were cBTKi therapy (47.7%), bendamustine-based therapy (17.0%), and venetoclax-based therapy (13.1%). Patients aged <75 years (odds ratio [OR] [95% CI]: 2.0 [1.2, 3.4]) and those who did not receive blood transfusion during cBTKi treatment (OR [95% CI]: 2.3 [1.3, 4.1]) were more likely to receive post-first-cBTKi treatment. In conclusion, Japanese patients with CLL/SLL received various treatments for short duration after first-cBTKi discontinuation.	[ { "section_content": "Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are hematological malignancies that are distinguishable from each other by their leukemic expression. 1,2In Japan, the annual age-adjusted incidence of CLL/ SLL has increased from 0.05-0.12 in 1993 to 0.12-0.27 in 2008 (per 100,000 person-years), an annual percentage change of 0.2%. 3,4he updated Japanese society of Hematology (JSH) guidelines and the International Workshop on Chronic Lymphocytic Leukemia guidelines for CLL recommended a \"watch and wait\" approach initially for patients without active CLL/SLL. 5,6According to the JSH guidelines, allogeneic stem cell transplantation is the only known curative treatment for patients with CLL/SLL who are resistant to molecular targeted therapies including covalent Bruton tyrosine kinase inhibitor (cBTKi) and B-cell lymphoma 2 inhibitor (BCL2i).However, it has limited use among patients with CLL/SLL who are generally older and have comorbidi-ties.For patients with active CLL/SLL in Japan, the available treatment options include chemoimmunotherapy, BCL2i, and cBTKi therapy. 5,7,8In first-line (1L) therapy, the JSH guidelines recommend treatment with the chemoimmunotherapeutic regimens of fludarabine, cyclophosphamide plus rituximab and bendamustine plus rituximab and ibrutinib (a cBTKi) monotherapy. 50][11][12] Treatment options after discontinuing cBTKi in either the first-line (1L) or for treatment of r/r CLL/SLL include venetoclax plus rituximab or chemoimmunotherapy. 5The selection of one treatment strategy over another is influenced by patients' age, comorbidities, molecular markers (immunoglobulin heavy chain gene, serum b2-microglobulin, del (17p), or TP53 mutations), patient preference, and treatment setting. 5,13Although patients with CLL/SLL are treated with different therapies after first-cBTKi discontinuation, no standard of treatment has been established for these patients. 5he primary aim of this study was to describe the patient characteristics, treatment patterns, and factors associated with receiving additional treatment in Japanese patients with CLL/ SLL who had received cBTKi therapy. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "This retrospective study used data from the Medical Data Vision Co., Ltd.(MDV) database (Tokyo, Japan) that were captured between April 2008 and February 2022.MDV is a de-identified inpatient and outpatient database of claims and diagnosis procedure combination data from acute care hospitals in Japan.A summary of the study design is included in Figure 1.Index date was defined as the date on which patients received their first treatment for CLL/SLL.Eligible patients received their index treatment between March 01, 2013, and February 28, 2021, (index period).As of February 01, 2022, the MDV database included approximately 39.6 million patients from 464 acute-phase hospitals in Japan (i.e., approximately 26% of acute hospitals).Of the 464 hospitals from which patients were enrolled in this study, 262 were designated cancer care hospitals (which provide specialized cancer care to the patients).These hospitals (classified by prefecture, community, or local hospitals) are designated by the Japan Ministry of Health, Labor, Family and Welfare.In addition to providing specialized treatments and care to patients with cancer, these hospitals co-ordinate and cooperate with each other and also act as information centers for the patients. 14his study was conducted in accordance with the ethical principles of the Declaration of Helsinki and Good Pharmacoepidemiology Practices.All personal information data were de-identified by each hospital and the database did not include the location of hospital, in compliance with the Act on the Protection of Personal Information in Japan.As this study collected de-identified retrospective data, the Japanese ethical guidelines for medical and health research involving human subjects did not apply; therefore, the study was exempted from the requirement for the ethical review and informed consent. ", "section_name": "Study design", "section_num": null }, { "section_content": "Patients aged ≥18 years (at diagnosis) and with confirmed diagnosis of CLL or SLL (International Classification of Diseases 10th Revision [ICD-10] World Health Organization code C83.0 for SLL, or C91.1 for CLL) during the index period, were identified (Figure 1).Among these patients, those who received one or more anti-neoplastic drug indicated for CLL or SLL (defined as CLL drugs, Supplementary Table S1) on or after the confirmed diagnosis of CLL or SLL during the index period were included. Patients were excluded if they had: 1) a diagnosis of CLL/SLL before March 01, 2013, or received any CLL drugs or stem cell transplantation prior to the study index period; 2) a confirmed diagnosis of Richter's transformation (ICD-10: C83.3, C81) throughout the study period; 3) been hospitalized as part of a clinical trial on or after diagnosis. For this analysis and report, the patients were grouped as those who received 1L treatment (1L cohort, overall), cBTKi treatment for the first time in any line (first-cBTKi cohort), and any treatment after the first-cBTKi treatment (post-first-cBTKi cohort). ", "section_name": "Patient population", "section_num": null }, { "section_content": "", "section_name": "Study outcomes", "section_num": null }, { "section_content": "", "section_name": "Primary outcomes", "section_num": null }, { "section_content": "Demographic and clinical characteristics were described during the baseline period (defined as 90 days before 1L) and included age, weight, body mass index, Charlson comorbidities index (CCI), 15 and 10-item Barthel activities of daily living (ADL) scale. 16These parameters were also described at initiation of first-cBTKi and post-first-cBTKi treatment.CCI measures the prognostic burden of comorbidities for predicting mortality, 15 which was calculated as the total number of predefined comorbid conditions identifiable in the claims based on ICD-10 codes.The Barthel Index ADL scale measures a patient's ability to perform daily activities and scores each question on a scale of 0 to 1, 0 to 2, or 0 to 3. 16 Total ADL was defined to be independent if all 10 items were recorded as independent, and not independent if any items were recorded as not independent.Total ADL was collected at each hospitalization.For patients with any ADL items unknown or missing, ADL was defined as missing.CCI was assessed at baseline and first-cBTKi treatment initiation.Hospital information including hospital size (classified by number of beds) and hospital type (designated cancer hospital or not) were described. ", "section_name": "Patient and hospital characteristics", "section_num": null }, { "section_content": "The sequence of treatment with 1L CLL drugs for each patient was described based on the groups presented in Supplementary Table S2.For grouping, the therapy base drug (base drug in a regimen) was given higher priority over other drugs in the group.Broadly, the groups were: i) Rituximab monotherapy; ii) cBTKi-based therapy; iii) Venetoclax-based therapy; iv) bendamustine-based therapy; v) fludarabine-based therapy; vi) doxorubicin-based therapy; vii) cyclophosphamide-based therapy; and viii) Other. All CLL drugs prescribed within the first 30 days following the index date constituted the 1L regimen.The treatment patterns where first-cBTKi was first prescribed and the immediate subsequent line (post-first-BTKi) treatment were assessed. ", "section_name": "Treatment patterns", "section_num": null }, { "section_content": "", "section_name": "Secondary outcomes", "section_num": null }, { "section_content": "Time to treatment discontinuation (TTD) was defined as the number of days between the start and end dates of the post-first-cBTKi line of treatment.Patients were considered to have discontinued the line of treatment if the interval between the end date of the line, the start of next line, and the end of hospital data was ≥90 days (date of the last prescription plus days of supply -1 day is defined as the end date of line), or the patient received another treatment for CLL (subsequent line of treatment; the line ends on the day before the introduction of subsequent treatment).Patients without discontinuation based on this definition were censored at the end date of the line.This was because such patients were likely to be on treatment on the last visit. ", "section_name": "Time to treatment discontinuation", "section_num": null }, { "section_content": "Factors associated with initiation of post-first-cBTKi treatment, assessed at first-cBTKi discontinuation, included presence or absence of atrial fibrillation, anthracycline treatment, anti-infective treatment, or congestive heart failure, receipt of blood transfusion (yes or no), hospital admission (yes or no), cBTKi use in 1 st line vs. 2 nd or later line), sex (male vs. female), age at cBTKi discontinuation (≥75 vs. <75 years), and total ADL score at discontinuation (independent vs. dependent). ", "section_name": "Factors associated with receipt of post-first-cBTKi treatment", "section_num": null }, { "section_content": "Descriptive statistics were used to present the hospital and patient characteristics for patients who received 1L therapy, patients who received first-cBTKi therapy, and patients who received post-first-cBTKi therapy.Characteristics of patients who discontinued first-cBTKi were also presented for those who received post-first-cBTKi therapy and for those in the first-cBTKi cohort who did not receive subsequent therapy.Categorical variables were presented as n (%) and continuous variables were presented as mean and standard deviation (SD) or median with interquartile range (IQR).TTD was calculated by Kaplan-Meier method.Imputation of missing data was not performed.Multivariable logistic regression analysis was used to explore factors associated with receiving post-first-cBTKi treatment.The multivariable logistic regression model with the same covariates mentioned above and ADL dependence (dependent vs. independent) was also used to explore the factors that may be associated with initiation of post-first-cBTKi treatment in subgroup of patients who had ADL data available at first-cBTKi discontinuation. All statistical analyses were conducted using the Instant Health Data software (Panalgo, Boston MA, USA) and R version 3.2.1 (R Foundation for Statistical Computing, Vienna, Austria). ", "section_name": "Statistical analyses", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "A total of 6,502 patients were diagnosed with CLL/SLL, of which 2,424 patients received 1L systemic therapy and were included in this study (overall population): 450 (18.6%) of eligible patients received cBTKi treatment in any line (Figure 2).Overall, patients had a median age of 72.0 years (IQR=65.0,79.0) and 61.9% were male.Patients had a mean CCI score of 2.2 (SD 1.4) (Table 1).The baseline characteristics among those who received first-cBTKi (n = 450) and post-first-cBTKi therapy (n = 153) are reported in Table 1. At 1L, 86.0% of patients received treatment at designated cancer hospitals and 56.4% of the patients were in hospitals with >500 beds.For first-cBTKi cohort and post-first-cBTKi cohort, proportion of receiving treatment at designated cancer hospitals (89.3% and 90.9%) or hospitals with >500 beds (60.4% and 59.4%) was higher (Table 1). ", "section_name": "Patient demographics and clinical and hospital characteristics", "section_num": null }, { "section_content": "Out of the 450 patients who received first-cBTKi treatment, 273 (60.7%) discontinued first-cBTKi treatment, 153 (34.0%) patients received post-BTKi treatment, and 120 (26.7%) patients did not receive post-first-cBTKi treatment (Figure 2 and Table 2).Also, a greater proportion of patients who did not receive post-first-cBTKi treatment were aged ≥75 years (57.5% vs. 41.8%,P = 0.014), used anti-infective drugs (35.0% vs. 20.9%,P = 0.014), emergent hospital admissions (24.2% vs. 13.1%,P = 0.027), and received at least one blood transfusion (41.7% vs. 21.6%,P = 0.001) during first-cBTKi treatment as compared with patients who received post-first-cBTKi treatment.At cBTKi discontinuation, proportion of patients with independent ADL was greater in patients with post-first-cBTKi than in without postfirst-cBTKi therapy (78.3% vs. 45.5%,P = 0.001) (Table 2). ", "section_name": "Patient characteristics after first-cBTKi discontinuation", "section_num": null }, { "section_content": "The most common treatment regimens in overall population (1L, n = 2,424) were bendamustine-based therapy (n = 668, 27.6%), fludarabine-based therapy (n = 413, 17.0%), cyclophosphamide-based therapy (n = 387, 16.0%), and rituximab monotherapy (n = 329, 13.6%).cBTKi was administered in 9.7% patients in 1L (n = 236/2,424), 13.5% patients in 2L (n = 142/1,055), and 14.1% patients in 3L (n = 70/491) (Figure 3). Among patients who received cBTKi therapy (n = 450), 54.2%, 23.6%, and 22.2% patients received first-cBTKi in 1L, 2L, and 3L or later, respectively. ", "section_name": "Treatment patterns", "section_num": null }, { "section_content": "In patients receiving therapy post-first-cBTKi (n = 153), the most common treatment regimens were cBTKi therapy (47.7%), bendamustine-based therapy (16.9%), venetoclaxbased therapy (13.1%), fludarabine-based therapy (6.5%) and rituximab monotherapy (3.3%) (Figure 4).The median Fig. 2. Study population selection and distribution duration of post-first-cBTKi treatment was 2.2 months (95% CI: 1.8, 3.5) (Figure 5).Among the post-first-cBTKi therapies, the median TTD was 3.5 months (95% CI: 2.0, 11.2) for cBTKi monotherapy and venetoclax-based therapy, 2.1 months (95% CI: 1.1, 4.0) and for bendamustine-based therapies, and 2.1 months (95% CI 0.0, not applicable [NA]) for doxorubicin-based therapies (Table 3). Supplementary Figures S1A andS1B present the treatment patterns in patients for whom cBTKi therapy ended before November 22, 2019 (i.e., venetoclax approval in Japan).In both the time periods, cBTKi therapy remained the preferred post-first-cBTKi therapy in patients with CLL, with the number of patients treated with venetoclax increasing in patients who ended first-cBTKi on or after November 22, 2019. ", "section_name": "Post-first-cBTKi treatment regimens and duration", "section_num": null }, { "section_content": "Factors associated with receipt of post-first-cBTKi therapy included age <75 years (odds ratio [OR]: 2.0 [95% CI 1.2, 3.4], P = 0.007) and lack of blood transfusion during first-cBTKi treatment (OR: 2.3 [95% CI 1.3, 4.1], P = 0.003) (Figure 6). In the subgroup of patients with ADL data available at first-cBTKi discontinuation (n = 113), receipt of post-first-cBTKi treatment was associated with age <75 years (OR: 3.8 [95% CI 1.5, 10.4], P = 0.007), female sex (OR: 3.0 [95% CI 1.1, 9.2], P = 0.039) and independent total ADL (OR: 3.5 [95% CI 1.4, 9.4], P = 0.009) (Supplementary Figure S2). ", "section_name": "Factors associated with receipt of post-first-cBTKi treatment", "section_num": null }, { "section_content": "In Japan, CLL/SLL is a rare disease. 3,17No standard treatment is established in patients with CLL/SLL after discontinuation of first-cBTKi and available treatment options are also limited.To the best of our knowledge, this is currently the largest study to describe the demographic and clinical characteristics at baseline and at first-cBTKi discontinuation, treatment patterns, and factors associated with receiving post-first-cBTKi treatment in Japanese patients with CLL/ SLL.Approximately 20% of the patients who received systemic therapy in this study were treated with at least one cBTKi.Among patients who received first-cBTKi treatment, more than 50% patients received subsequent treatment after first-cBTKi treatment discontinuation, but for a short duration. At 1L treatment, median age of patients (72.0 years) and burden of comorbidities (median CCI = 2.2) in the current study were similar to the median age and CCI reported in a previous real-world study of an earlier cohort of Japanese patients with CLL in the MDV between March 2013 and February 2018 (median age = 70.1 years; median CCI = 1L: first-line; ADL: activities of daily living; BMI: body mass index; cBTKi: covalent Bruton's tyrosine kinase inhibitor; CCI: Charlson comorbidities index; IQR: interquartile range; SD: standard deviation a Weights were only available from patient discharge for 1,802 (74.3%) patients in 1L cohort, 241 (53.5%) in cBTKi cohort, and 100 (65.4%) in post-cBTKi cohort.b BMI were available only from patient discharge summaries for 1,815 (74.9%) patients in 1L cohort; 240 (53.3%) n cBTKi cohort; 99 (64.7%) in post-first-cBTKi cohort.C Data on ADL at discontinuation were available only from patient discharge summaries for 1,720 (71.0%) patients in 1L cohort, 223 (49.6%) patients in cBTKi cohort, and 92 (60.1%) patients in post-first-cBTKi cohort. Baseline was defined as 90 days period before initiation of 1L, first-cBTKi treatment, and post-first -cBTKi treatment lines.2.2). 18 In the present study, patients aged <75 years, those not receiving blood transfusion, and those independently performing ADL were more likely to receive post-first-cBTKi treatment.Previous studies have also demonstrated that older age and comorbidities adversely impact the treatment outcomes in patients with CLL. 19,20Hence, advanced age (≥75 years) and increased comorbidities in the current study post-first-cBTKi treatment may indicate poor prognosis of CLL/SLL, with no treatment received post-first-cBTKi. The most prescribed 1L therapies were bendamustinebased regimens (27.6%), fludarabine-based regimens (17.0%), cyclophosphamide-based regimens (16.0%), and rituximab monotherapy (13.6%).In the CLIMBER-DBR study, discussed previously, fludarabine-based (17.7%) and cyclophosphamide-based (13.7%) regimens were the main 1L treatments for CLL, with cBTKi administered in 2.6% patients. 18The difference in 1L treatment patterns in the current study versus the previous study could be due to the availability of newer drugs for treatment of CLL/SLL in the current study given the differences in time periods investigated. The primary outcome of this study was to assess treatment patterns post-first-cBTKi therapy.In the current study, 18.6% (450/2,424) of patients received at least one cBTKi treatment.Among the patients who received post-first-cBTKi treatment for CLL, cBTKi monotherapy (47.7%), bendamustine-based regimen (16.9%) and venetoclax-based regimen (13.1%) were the most common post-first-cBTKi treatment regimens.cBTKi are associated with multiple toxicities including cardiac and cardiovascular events, arthralgia, infections, diarrhea, dermatological manifestations, headache. 21After discontinuation of first-cBTKi due to disease progression, 22 potential to switch to another cBTKi is low due to the risk of cross-resistance between different cBTKi. 23o, majority of patients who received cBTKi as post-first-cBTKi therapy may discontinue or interrupt their first-cBTKi because of AEs and a proportion of patients continue with the same cBTKi after recovering from AEs.This is consistent with our study where all 73 patients treated with post-first cBTKi had received the same cBTKi regimen as received in ADL: Activity of daily living; cBTKi: covalent Bruton's tyrosine kinase inhibitor; CCI: Charlson comorbidities index; SD: Standard deviation Baseline was defined as the 90-day period before initiation of 1L.Newly emerged outcomes are defined as yes when the corresponding diagnosis codes were found during the first-cBTKi treatment but not shown in the baseline of (90 days prior to) the cBTKi therapy.Median (95% CI) duration of first-cBTKi was calculated by Kaplan-Meier method.a Data on ADL at baseline were available only from patient discharge summaries for 64 (53.3%) patients in No Post-cBTKi cohort, 83 (54.3%) patients in With Post-first-cBTKi cohort.b Data on ADL at cBTKi discontinuation were available only from patient discharge summaries for 44 (36.7%) patients in No Post-cBTKi cohort; 69 (45.1%) in With Post-first-cBTKi cohort.Categorical variables analyzed using Chi2 test and Continuous variables analyzed using student t-test.the previous line.In the CLIMBER-DBR study, 2.6% patients received cBTKi in 1L, 10.5% patients received cBTKi in 2L, and 13.8% received cBTKi in 3L, 18 which was lower than the proportion pf patients receiving cBTKi therapy across 1L-3L in our study (9.7% in 1L, 13.5% in 2L, and 14.1%), likely due to the timing of the study, which was conducted between 2013 and 2018 (ibrutinib was approved in Japan in 2016 and acalabrutinib in 2021). 18,24,25Therefore, the current body of evidence is valuable as it adds insights into the treatment patterns of this small subset of patient population in a more contemporaneous cohort of Japanese patients with CLL/SLL.In this study, the median duration of the immediate postfirst-cBTKi treatment was short (2.2 months), suggesting there is a need for more effective therapies among patients with CLL/SLL who require treatment post-first-cBTKi.The S2.Median (95% CI) TTD was calculated by Kaplan-Meier method.frequently used post-first-cBTKi treatments used in the current study (e.g., bendamustine, rituximab) have a fixed treatment durations (up to 6 months). 26,27In this study, we analyzed the treatment duration of each regimen and found that median TTD of bendamustine-based treatment was 2.1 months (95% CI: 1.1, 4.0) and rituximab monotherapy was 1.3 months (95% CI: 0.8, NA).These results suggested that fixed duration may not be the only reason for a shorter TTD of post-first-cBTKi treatments.Other reasons could be their inadequate efficacy or safety. 21Temporary discontinuation of post-first-cBTKi treatments to manage adverse events could be another reason for short TTD of these treatments. 26,27ewer, second-generation, cBTKi agents (e.g.acalabrutinib) are approved for CLL as monotherapy. 24,25Noncovalent BTKi therapy (pirtobrutinib) 28 is under investigation for patients with r/r CLL. 29New combination therapies (acalabrutinib plus obinutuzumab) are also under investigation for treatment of CLL. 30 The second-generation cBTKi drugs have greater selectivity compared with first-generation cBTKi drugs; however, they fail to overcome resistance induced by BTK mutations. 31,32Venetoclax, a BCL2i, is an approved agent that has demonstrated favorable efficacy (65% overall response rate) in patients with r/r CLL who progressed on or discontinued ibrutinib. 33Based on the short durations of post-first-cBTKi therapy, there is a clear need for effective and safe therapies after discontinuation of cBTKi therapy. ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "This study used the MDV database, which includes large number of patients who received care in a hospital setting.Although the database has large number of patients which enables the study of a relatively rare condition such as CLL, it also has inherent limitations which limit the generalizability of this study.The MDV database does not incorporate information from facilities outside the database contract or from non-hospital care.Therefore, data for patients treated at multiple facilities may be incomplete or duplicative.In addition, reasons for treatment delay or discontinuation were also not available in the database.Hence, temporary interruption of treatment for ≥90 days could have been counted as treatment discontinuation and start of new line.Reason for discontinuation of cBTKi was not known.Lastly, short treatment duration (~2 months) could be due to inadequate efficacy, safety, temporary discontinuation, or fixed treatment duration of some post-first-cBTKi treatments used in the current study (e.g.bendamustine and rituximab). 34,35 ", "section_name": "Strengths and limitations", "section_num": null }, { "section_content": "This real-world database study in Japan demonstrated that more than half of patients with CLL/SLL who discontinued first-cBTKi treatment received post-first-cBTKi treatment for a short duration (median 2.2 months).Overall, age <75 years and no history of blood transfusion increased the probability of receiving post-first-cBTKi treatment in the patient population.For patients with available ADL data, age <75 years, female sex, and ADL independence were associated with increased likelihood to receive post-first-cBTKi treatment.These data suggest an unmet medical need and lack of standardized treatment for patients with CLL/SLL after discontinuation of first-cBTKi treatment. ", "section_name": "CONCLUSIONS", "section_num": null } ]	[ { "section_content": "Medical writing support under the guidance of the authors was provided by Karan Sharma and Rahul Nikam from Eli Lilly Services India Pvt. Ltd., which was funded by Eli Lilly and Company.Editorial and submission support was provided by Leo J. Philip Tharappel from Eli Lilly Services India Pvt. Ltd. ", "section_name": "ACKNOWLEDGMENTS", "section_num": null }, { "section_content": "This study was sponsored by Eli Lilly and Company. ", "section_name": "FUNDING", "section_num": null }, { "section_content": "All authors made a significant contribution to the work reported whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for the content of the article. Dai Maruyama is an employee of Cancer Institute Hospital, Japanese Foundation for Cancer Research.Shigeru Kusumoto is an employee of Aichi Cancer Center Hospital.Previously, he was employed by Nagoya City University Graduate School of Medical Sciences.Chaochen Wang, Yoshinori Tanizawa, Zhihong Cai, and Masaomi Tajimi are full-time employees of Eli Lilly Japan K.K. Yu-Jing Huang is a full-time employee of Eli Lilly and Company.Chaochen Wang, Yoshinori Tanizawa, Zhihong Cai, Masaomi Tajimi, and Yu-Jing Huang are minor stockholders of Eli Lilly and Company. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "All authors made a significant contribution to the work reported whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for the content of the article. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "Dai Maruyama is an employee of Cancer Institute Hospital, Japanese Foundation for Cancer Research.Shigeru Kusumoto is an employee of Aichi Cancer Center Hospital.Previously, he was employed by Nagoya City University Graduate School of Medical Sciences.Chaochen Wang, Yoshinori Tanizawa, Zhihong Cai, and Masaomi Tajimi are full-time employees of Eli Lilly Japan K.K. Yu-Jing Huang is a full-time employee of Eli Lilly and Company.Chaochen Wang, Yoshinori Tanizawa, Zhihong Cai, Masaomi Tajimi, and Yu-Jing Huang are minor stockholders of Eli Lilly and Company. ", "section_name": "CONFLICT OF INTEREST", "section_num": null } ]
10.3389/fnins.2023.1163806	Evaluation of dihydrotestosterone and dihydroprogesterone levels and gene expression of genes involved in neurosteroidogenesis in the SH-SY5Y Alzheimer disease cell model	<jats:sec><jats:title>Introduction</jats:title><jats:p>Alzheimer’s disease (AD) is the most common form of dementia worldwide. This study investigated the effects of lipopolysaccharide on neurosteroidogenesis and its relationship to growth and differentiation using SH-SY5Y cells.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>In this study, we used the MTT assay to assess the impact of LPS on SH-SY5Y cell viability. We also evaluated apoptotic effects using FITC Annexin V staining to detect phosphatidylserine in the cell membrane. To identify gene expression related to human neurogenesis, we utilized the RT<jats:sup>2</jats:sup> Profiler TM PCR array human neurogenesis PAHS-404Z.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Our study found that LPS had an IC50 level of 0.25 μg/mL on the SH-SY5Y cell line after 48 h. We observed Aβ deposition in SH-SY5Y cells treated with LPS, and a decrease in DHT and DHP levels in the cells. Our analysis showed that the total rate of apoptosis varied with LPS dilution: 4.6% at 0.1 μg/mL, 10.5% at 10 μg/mL, and 44.1% at 50 μg/mL. We also observed an increase in the expression of several genes involved in human neurogenesis, including ASCL1, BCL2, BDNF, CDK5R1, CDK5RAP2, CREB1, DRD2, HES1, HEYL, NOTCH1, STAT3, and TGFB1, after treatment with LPS at 10 μg/mL and 50 μg/mL. LPS at 50 μg/mL increased the expression of FLNA and NEUROG2, as well as the other genes mentioned.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>Our study showed that LPS treatment altered the expression of human neurogenesis genes and decreased DHT and DHP levels in SH-SY5Y cells. These findings suggest that targeting LPS, DHT, and DHP could be potential therapeutic strategies to treat AD or improve its symptoms.</jats:p></jats:sec>	[ { "section_content": "The expression of Aβ, tau, synaptic factors, and other neuronspecific proteins in SH-SY5Y cells makes them a suitable model to study the mechanism of neuron phenotype degeneration, including AD (Aitken et al., 2016).AD is a neurological disorder and the most common cause of dementia in older adults (Hoseinlar et al., 2023;Shademan et al., 2023).Although more common in women, it can occur in both men and women.Statistically, there are estimated 5.2 million people in the United States with the disease, including 3.3 million women and 1.9 million men (Aslanpour et al., 2020a).Several studies have been conducted to treat and prevent AD, but a definitive treatment has not yet been approved.Drug treatment and medical costs are three times higher for people with Alzheimer's disease than for others.Factors such as immobility, high blood sugar, cholesterol levels, and genetic history can play a role in the development of AD (Aslanpour et al., 2020b).The neuropathology of Alzheimer's disease is mainly affected by extracellular accumulation of amyloid beta (Aβ), accumulation of tau in neurons, glial activation, and loss of neurons and synapses (Hoseinlar et al., 2023).Neuropathological changes are associated with glial activity that causes nerve damage, loss of synapses, and neuronal death (Lawrence et al., 2023).Although the mechanisms underlying AD pathogenesis are not fully understood, amyloid plaques are thought to be involved in disease progression.The primary precursors for forming Aβ-plaques are the amyloid beta protein and neurofibrillary tau tangles in the brain.The primary amyloid precursor performs many functions, from neurotransmission to gene transcription (Beydoun et al., 2021). Pregnant mice exposed to repeated systemic exposure to LPS (lipopolysaccharide) cause Alzheimer's disease-related features, including behavioral and neuropathological changes, in their offspring (Wang et al., 2020).Despite the limited and temporary induction of neuronal damage, a single systemic challenge of LPS (lipopolysaccharide) leads to increased deposition of Aβ1-42 and tau levels in the brains of wild-type rodents (Wang et al., 2018).Moreover, repeated systemic injections of LPS (lipopolysaccharide) can lead to prolonged elevation of Aβ levels and cognitive deficits (Xie et al., 2022). Sex hormones can influence growth, synaptogenesis, dendritic branching, and myelination (Calan et al., 2016), and other important mechanisms of neuroplasticity (Calan et al., 2016).Sex hormones are released into various tissues and the brain via the bloodstream.In addition to the sex glands, they are also produced in the brain and can improve brain function (Carroll et al., 2010).Sex hormones can regulate neuronal survival in different areas of the central nervous system (CNS) and promote repair of neuronal damage (Castro et al., 2011).Sex hormones can affect different areas of the CNS, including the brain, spinal cord, and peripheral nerves, due to the presence of estrogen and progesterone receptors in these sites.The increased risk of AD is significantly influenced by sexual steroid hormones in women.Decreased levels of progesterone are associated with an increased risk of AD.In men, the risk of developing AD is also affected by the male sex steroid hormone testosterone.The rate of testosterone production gradually decreases with age, leading to an increased risk of developing AD (Radaghdam et al., 2021).There is still debate about whether sex hormones should be used as a treatment or prevention strategy for diseases associated with neurological damage, including AD.However, progesterone and testosterone can reduce AD in several ways, including the MAPK/ERK and CREB pathways (Chouchane and Costa, 2019). LPS exposure can lead to various chronic diseases and induce mitochondrial apoptosis in neurodegenerative conditions.Sirtuin 1 plays a key role in the function of testosterone and estrogen receptors, along with androgens and estrogens.The concentration of LPS may affect the expression of the Sirtuin 1 gene, which in turn may be related to the production of dihydrotestosterone (DHT) and dihydroprogesterone (DHP), as well as neurogenesis.Therefore, the effect of LPS concentration on gene expression may be associated with DHT and DHP (Ian, 2017;Martins, 2018;Rasha et al., 2020;Tsuchiya et al., 2020).Identification of the genes involved in these mechanisms can aid in the development of treatments and management strategies for AD. Examination of changes in the expression of genes related to human neuroblastoma neurogenesis in SH-SY5Y cells under the influence of LPS may offer new information on the submechanisms involved.This study aimed to investigate the effect of lipopolysaccharide (LPS) on neurosteroidogenesis and its relationship with growth and differentiation, as well as the expression patterns of genes involved in neurogenesis in the human neuroblastoma cell line, SH-SY5Y. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "", "section_name": "Materials and methods", "section_num": "2." }, { "section_content": "The SH-SY5Y cell line was obtained from the cell bank of the Pasteur Institute (Tehran, Iran).The cells were cultured in Dulbecco's modified Eagle Medium/Nutrient Mixture F-12 medium (DMEM-F12, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin (100 units/mL, Biosera), streptomycin (100 μg/mL, Biosera), and 250 μmol/L cholesterol.The cells were incubated at 37°C with 95% humidity and 5% CO 2 .After 48 h, cells were harvested at 70-80% confluence for further analysis.To prepare a concentration of 250 μmol/L cholesterol, we dissolved 9.6 mg of cholesterol in 1 cc of ethanol and diluted at 1:100 with RPMI medium.The cells were then treated with different concentrations of LPS, including 0.1, 10, and 50 μg/mL (as a control group) for 24, 48, and 72 h, respectively.Based on the results of the MTT assay, we selected LPS 10 μg/mL for subsequent analyses. ", "section_name": "Cell line, culture conditions, and chemicals", "section_num": "2.1." }, { "section_content": "We used the MTT assay (Gibco, United States) to assess the effect of LPS (Chen and Song, 2020) on cell viability in SH-SY5Y cells.SH-SY5Y cells were seeded in 96-well plates at a density of 1 × 104 cells/well and incubated for 24 h under standard conditions.The cells were then treated with different concentrations of LPS (0.1, 10, and 50 μg/mL) for 24, 48, and 72 h.The medium was replaced with 100 μL of MTT solution (5 mg/mL in PBS) and incubated for 4 h under standard conditions.Subsequently, 50 μL of dimethyl sulfoxide (DMSO; Merck) was added to each well and the plates were incubated for 30 min at 37°C.LPS IC50 values for LPS in the SH-SY5Y cell line were determined after 48 h, and subsequent experiments were performed using this concentration.The optical density (OD) of the wells was measured at 570 nm using a microplate reader (BioTek, United States), and cell viability was evaluated based on the results. ", "section_name": "Cell viability assay", "section_num": "2.2." }, { "section_content": "The control group (SH-SY5Y cells cultured in a medium without cholesterol and LPS) and the case group (treated with LPS) were used to examine amyloid beta deposits with Congo staining.Amyloid detection was performed according to the instructions of the Vitro View Congo Red Amyloid Stain Kit (Biotech No. VB-3011, SKU).The CKX53 inverted microscope (Olympus) with a built-in camera port was used to detect amyloid deposits.When amyloid deposits were present in the environment, they became visible in red, while the nuclei appeared blue. ", "section_name": "Deposition of amyloid beta", "section_num": "2.3." }, { "section_content": "DHT (Cat.No: MBS762135, MyBioSource, United States) and DHP (Cat No: DElA1592, Creative Diagnostics, United States), were quantified by the competitive ELISA detection method according to the manufacturer's instructions.For this purpose, 1 × 10 4 cells/well were plated in 96-well plates and treated with different concentrations of LPS (0.1, 10, and 50 μg/mL).Cell culture supernatant was centrifuged at 1000 × g and 2-8°C for 20 min to remove insoluble contaminants and cell debris.We diluted the buffer with a protease inhibitor (1:2) to prevent protein degradation.The OD was measured using a microplate reader (BioTek, United States) at a wavelength of 450 nm. ", "section_name": "Measurement of dihydrotestosterone and dihydroprogesterone in SH-SY5Y cell line", "section_num": "2.4." }, { "section_content": "The apoptotic effects of the drugs on the cell lines based on phosphatidylserine in the cell membrane were evaluated using the FITC Annexin V apoptosis detection method, following the protocol.For this purpose, 1 × 10 4 cells/well were counted on the BD ACCURI C6 flow cytometer (BD Biosciences Pharmingen).The data obtained from flow cytometry were analyzed using FlowJo software version 7.6.1.The apoptotic effects of the drugs were examined 48 h after they were added to cells at the indicated doses.The groups that were not treated with the drugs served as controls. ", "section_name": "Evaluation of apoptosis", "section_num": "2.5." }, { "section_content": "SH-SY5Y cells were seeded in a 6-well plate (1 × 10 4 cells/well) and incubated for 24 h under standard conditions.The expression changes induced by the IC50 doses of LPS in the SH-SY5Y cell line were determined using real-time polymerase chain reaction for 48 h.Total RNA was obtained from SH-SY5Y cells using the RNeasy kit (Cat.No.: FABRK001, Taiwan).Complementary DNA synthesis was performed using an RT2 first-strand kit (Cat.No.: YT4500, Austria).Changes in the expression of 96 genes associated with neurogenesis were examined using the RT2 Profiler TM Human Neurogenesis PCR Array (Qiagen, Cat.No.: PAHS-404Z) and the Light Cycler 480 instrument II (Roche).Data were analyzed using the comparative 2 - ΔΔCT method (Light Cycler 480 Quantification Software) using the changes made.Differences of more than ±2-fold change in expression were considered cut-off values with p-values <0.05. ", "section_name": "Determination of gene expression changes", "section_num": "2.6." }, { "section_content": "Statistical analyses were performed using SPSS version 16.Differences between the treated groups and control groups were analyzed with one-way ANOVA, followed by Tukey's post hoc analyses.All variables are expressed as mean ± SD.Statistical significance was established at p < 0.05. ", "section_name": "Statistical analysis", "section_num": "2.7." }, { "section_content": "", "section_name": "Results", "section_num": "3." }, { "section_content": "To determine the value of LPS IC50, LPS dilutions of 0.1, 10, and 50 μg/mL were tested by the MTT assay at 24, 48, and 72 h in the SH-SY5Y cell line.The IC50 values of LPS for the SH-SY5Y cell line were 0.25 μg/mL at 48 h.Our results showed that cell death increased with increasing LPS dose (Figure 1). ", "section_name": "Evaluation of the effect of LPS on cell viability in SH-SY5Y cell line", "section_num": "3.1." }, { "section_content": "Congo red staining was performed to investigate Aβ deposition.The results indicated that LPS increased Aβ toxicity in SH-SY5Y cells, as amyloid beta deposition was observed in the cells treated with LPS (Figure 2).LPS decreases DHT and DHP in SH-SY5Y cells.The ELISA results showed that as the LPS concentration increased, the concentrations of DHT and DHP decreased, with the maximum decrease in DHT and DHP concentrations observed at 50 μg/mL of LPS (p < 0.001) and a decrease in concentration observed at 0.1 μg/mL of LPS (p < 0.05) (Figure 3). ", "section_name": "Investigating the effect of LPS on the deposition of beta-amyloid", "section_num": "3.2." }, { "section_content": "Our study demonstrated that LPS-treated SH-SY5Y cells had significantly higher rates of early and late apoptosis than untreated cells.Furthermore, the total apoptosis rate in SH-SY5Y cells increased with increasing LPS concentration, with rates of 4.6, 10.5, and 44.1% observed for LPS dilutions of 0.1 μg/mL, 10 μg/mL, and 50 μg/mL, respectively.Notably, the overall apoptosis rate in SH-SY5Y cells treated with 50 μg/mL LPS was significantly higher than that of the other treatment groups (Figure 4). ", "section_name": "Evaluation of the effect of LPS on apoptosis in SH-SY5Y cell line", "section_num": "3.4." }, { "section_content": "We examined the effect of LPS on gene expression related to neurogenesis.The PCR array analysis indicated that LPS stimulates the expression of genes involved in human neuroblastoma neurogenesis (Table 1).LPS increased the expression of the ASCL1, BCL2, BDNF, NEUROG2, and PTEN genes was increased by 0.1 μg/ mL.Additionally, LPS increased the expression of ASCL1, BCL2, BDNF, CDK5R1, CDK5RAP2, CREB1, DRD2, HES1, HEYL, NOTCH1, STAT3, and TGFB1 by LPS 10 μg/mL.Finally, LPS 50 μg/ mL upregulated the expression levels of BCL2, BDNF, CDK5R1, CDK5RAP2, CREB1, DRD2, FLNA, NEUROG2, NOTCH1, PTEN, STAT3, and TGFB1 genes (Figure 5). ", "section_name": "Gene expression changes caused by LPS in SH-SY5Y cell line", "section_num": "3.5." }, { "section_content": "Previous studies have shown that LPS causes a decrease in synaptic plasticity, cognitive function, and neuronal function by damaging myelin in (AD) (Cosgrove et al., 2007;Congdon and Sigurdsson, 2018).Furthermore, affecting the inhibitory and excitatory synapses of hippocampal neurons causes loss of function in the CNS (Dai et al., 2021).As a significant ligand, LPS increases the expression of TLR4 and caspase-11 genes by targeting the neuronal TLR4 receptor.Consequently, it causes neuronal cell death in AD by activating the inflammatory response (Duhr et al., 2014;Fu et al., 2019).Because TLR4 expression increases with age and amyloid beta levels, the interaction between LPS and TLR4 could influence the development of AD (Fulop et al., 2018).Although the general mechanism of AD is not fully understood, the role of amyloid beta toxicity in the pathogenesis of AD is vital (Garcia-Ovejero et al., 2005).Our study showed that different concentrations of LPS can have cytotoxic effects on SH-SY5Y cells, and increased amyloid beta deposition can exacerbate apoptosis in SH-SY5Y cells. Cholesterol is essential as a basis for producing steroid hormones, including progesterone and estrogen, and these two hormones play various physiological functions in both men and women.Because of the critical role of progesterone and estrogen in neurodegenerative Quantification of apoptosis by Annexin V-FITC/propidium iodide (PI) assay in SH-SY5Y cells treated with LPS dilutions of 0.1, 10, and 50 μg/mL.The induction of apoptosis in SH-SY5Y cells treated with LPS 50 μg/mL was significantly higher than in other treatments.FITC, fluorescein isothiocyanate; PI.Clustergram analysis of genes involved in human neurogenesis after incubation with different concentrations of LPS in SH-SY5Y cells (n = 3).Here, the red color denotes the up regulation of genes across specific samples/conditions, and the green color denotes the downregulation of genes across the specific samples/conditions. diseases, including AD, many studies have investigated changes in these two hormones.Some studies have shown that the use of estrogen reduces the risk of AD in women.Therefore, estrogen compounds may be used to treat AD in the elderly (Gascón et al., 2016;Grothe et al., 2017).However, because hormone therapy is expensive and lengthy, patients often abandon treatment (He et al., 2014).Some studies have shown that testosterone and progesterone play an influential role in neuroprotection during the early stages of AD development.However, it is challenging to apply these two hormones effectively in the later phases of the disease (Heinrich et al., 2014;Huo et al., 2016;Hung et al., 2017;He et al., 2019).Furthermore, DHT has been found to modulate the expression of Aβ, the caspase-3, Bcl-2 and Bax, and synaptophysin, as well as reduce neuronal damage in mice treated with LPS.DHT also exerts anti-neuroinflammatory and neuroprotective effects, making androgen replacement therapy a potential therapeutic strategy for improving cognitive and behavioral function in neuroinflammation-related diseases (Yang et al., 2020). Our study revealed that an increase in LPS concentration led to a decrease in both DHT and DHP concentrations.The maximum decrease in DHT and DHP concentration occurred at an LPS concentration of 50 μg/mL.Previous research has shown that LPS in the hypothalamus or pituitary gland can disrupt follicular growth and function in mice by suppressing gonadotropin release (Kuo et al., 2010;Kitagishi and Matsuda, 2013;Johnson et al., 2016).Additionally, in follicles with high LPS compared to those with low LPS, there was a suppression of CYP17 gene expression in theca cells and P450 aromatase gene expression in granulosa cells, resulting in decreased estrogen levels (E2) (Lin et al., 2010;Lee et al., 2014;Liu et al., 2017).LPS has also been shown to reduce progesterone biosynthesis in mice (Mao and Sun, 2015;Masters et al., 2015).These findings suggest that menopause and the decrease in sex hormones, such as estrogen and progesterone in women and testosterone in men, may increase the susceptibility to AD in old age.0.1 μg/mL LPS increased the expression of ASCL1, BCL2, BDNF, NEUROG2, and PTEN.LPS (10 μg/mL) increased the expression of ASCL1, BCL2, BDNF, CDK5R1, CDK5RAP2, CREB1, DRD2, HES1, HEYL, NOTCH1, STAT3, and TGFB1.50 μg/mL LPS increased the expression levels of BCL2, BDNF, CDK5R1, CDK5RAP2, CREB1, DRD2, FLNA, NEUROG2, NOTCH1, PTEN, STAT3, and TGFB1.The results of our study show that LPS concentration has a differential effect on the expression of genes related to neurogenesis in human neuroblastoma.Increasing LPS concentration can significantly affect the expression of neurogenesis-related genes in human neuroblastoma.In AD, neuron destruction is triggered by increased CDK5 activity.CDK5 kinase, abundantly expressed in neurons and plays a critical role in synaptic plasticity and neuronal development, is implicated in triggering neuronal destruction in AD through increased activity (Pan et al., 2016;Park et al., 2021).While CDK5 overactivity is linked to the development of neurodegeneration, it also plays a crucial role in various physiological functions, including migration, neuroblasts, and synaptic plasticity.CDK5 is located at the end of the axon growth cone, where it regulates the growth of neural progenitor cells into mature neurons, making it necessary for the maturation phase of neurogenesis.Altered CDK5 activity in neural progenitor cells is associated with defects in neurogenesis in AD (Pascual et al., 2015).We also observed increased expression of genes, including FLNA and STAT3, both of which function as cofactors and transcription factors.Therefore, FLNA is considered associated with the pathogenesis of amyloid beta and tau proteins in AD (Pitsavos et al., 2006;Patricia et al., 2013).Amyloid beta induces the production of this protein, which plays a crucial role in the AD signaling pathway.Persistent activation of the TLR4 receptor by beta-amyloid causes an overproduction of inflammatory cytokines and triggers neuroinflammation (Pompili et al., 2012;Ruigrok et al., 2014;Robinson et al., 2016;Schöll et al., 2016).Furthermore, it is understandable that the limitations of cell lines in mimicking AD and the events that occur in AD make the field stronger for detailed studies in animal models (Zhan et al., 2018). ", "section_name": "Discussion", "section_num": "4." }, { "section_content": "Different concentrations of LPS can have cytotoxic effects on SH-SY5Y cells, and increasing amyloid beta deposition may enhance apoptosis in these cells.Moreover, LPS can reduce the concentration of DHT and DHP.Changes in the expression of genes related to neurogenesis in human neuroblastoma cells under the influence of LPS suggest novel sub-mechanisms.Targeting DHT and DHP or neurogenesis in human neuroblastoma cells may be a promising therapeutic strategy for AD treatment or symptom relief.However, further studies are needed to fully explain the underlying mechanism. ", "section_name": "Conclusion", "section_num": "5." } ]	[ { "section_content": "The authors appreciate the kind help of the Stem Cell Research Center of Tabriz University of Medical Sciences. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "This work was supported by a grant from the Vice Chancellor for Research, Tabriz University of Medical Sciences, Tabriz, Iran, with the grant number IR.TBZMED.REC.1396.896. ", "section_name": "Funding", "section_num": null }, { "section_content": "The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. ", "section_name": "Data availability statement", "section_num": null }, { "section_content": "The Ethics Committee of the Tabriz University of Medical Sciences approved the study protocols. AN and FK-K were involved in the study design.SN and BS contributed to the analysis and interpretation of data.BS, RR, and MN contributed to revising the manuscript content.AN gave consent for the final version.SR wrote the manuscript.All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. ", "section_name": "Ethics statement", "section_num": null }, { "section_content": "The Ethics Committee of the Tabriz University of Medical Sciences approved the study protocols. ", "section_name": "Ethics statement", "section_num": null }, { "section_content": "AN and FK-K were involved in the study design.SN and BS contributed to the analysis and interpretation of data.BS, RR, and MN contributed to revising the manuscript content.AN gave consent for the final version.SR wrote the manuscript.All authors contributed to the article and approved the submitted version. ", "section_name": "Author contributions", "section_num": null }, { "section_content": "The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ", "section_name": "Conflict of interest", "section_num": null }, { "section_content": "All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. ", "section_name": "Publisher's note", "section_num": null } ]
10.1186/s12885-016-2205-5	Distribution and features of hematological malignancies in Eastern Morocco: a retrospective multicenter study over 5 years	Las neoplasias hematológicas (HM) son un problema de salud pública. El patrón y la distribución de los cánceres hematológicos diagnosticados varían según la edad, el sexo, la geografía y el origen étnico, lo que sugiere la participación de factores genéticos y ambientales en el desarrollo de estas enfermedades. Hasta donde sabemos, no hay ningún informe publicado sobre HM en el caso del este de Marruecos. En este informe presentamos por primera vez el patrón general de HM para esta región. Estudio descriptivo retrospectivo de pacientes diagnosticados de MH entre enero de 2008 y diciembre de 2012 en tres centros del este de Marruecos que prestan servicios de diagnóstico, tratamiento o cuidados paliativos del cáncer. Se ha tenido en cuenta el sistema de clasificación FAB (franco-americano-británico) en el análisis de neoplasias mieloides y linfoides. En este estudio, se registraron un total de 660 casos de HM entre enero de 2008 y diciembre de 2012. En general, se registraron 6075 casos de cánceres en todos los sitios combinados durante este período de estudio, lo que indica que la HM representa alrededor del 10,9 % (660/6075) de todos los cánceres registrados. Entre los 660 casos registrados de HM, el 53% eran hombres y el 47% eran mujeres, con una proporción de hombres a mujeres de 1,1. Por lo tanto, en general, los hombres se ven ligeramente más afectados con HM que las mujeres. Por el contrario, se observó un predominio femenino en el caso del linfoma de Hodgkin (LH), las neoplasias mieloproliferativas (NMP), la leucemia mieloide aguda (LMA) y el síndrome mielodisplásico (SMD). La HM se produce a una edad relativamente temprana, con una mediana de edad general en el momento del diagnóstico de 54 años. El linfoma no Hodgkin (NHL) fue el HM más común y representó el 29,7 % de todos los HM, seguido del HL, MPN, mielomas múltiples (MM), leucemia linfocítica crónica (CLL), AML, MDS, leucemia linfoblástica aguda (ALL) y macroglobulinemia de Waldenström (WM). La mayoría de los casos de HM se han observado entre pacientes de 60 años o más (40,4 % de HM). Entre este grupo de edad, el LNH fue el HM más común. En adolescentes, HL fue la HM más frecuente. Este estudio proporcionó por primera vez el patrón y la distribución de HM en el este de Marruecos. Nuestros hallazgos justifican la necesidad de establecer un registro regional de cáncer como primer paso en el control del cáncer de sangre en el este de Marruecos.	[ { "section_content": "The HM are a group of cancers that arise from a malignant transformation of cells of the bone marrow or the lymphatic system [1].Several classification systems have been developed over the past several years to subdivide the HM by relevant clinical and biological means.These classifications facilitate the recognition of HM and therefore refine our ability to diagnose and treat these cancers.Moreover, they enable the use of the same definition of a specific hematopoietic neoplasm, so that data comparison between studies is possible [2].These classifications have been updated since the early 1970s, and in 2001 the World Health Organization (WHO) published a classification of tumours of the hematopoietic and lymphoid tissues.This 2001 WHO classification was the first worldwide consensus classification on hematological tumours and was based on multiple information, such as clinical, morphologic, biologic, immunophenotypic and genetic features [3,4].In 2008, the WHO published a new classification for hematopoietic and lymphoid neoplasms in conjunction with the Society for Hematopathology and the European Association of Hematopathology [3,5].This 4th edition of the WHO classification has refined the 2001 classification on the basis of new and cumulative experimental evidence [2,[5][6][7][8][9][10][11].It includes new criteria for the recognition of some previously described neoplasms and information about new entities that have been defined mainly by genetic mutations that have been characterized only recently [5,6,11]. There are two major groups of HM according to their cell lineage: myeloid and lymphoid [7,11].Lymphoid neoplasms are a very varied group and the common subgroups are: NHL, HL, MM, WM, ALL, and CLL.Myeloid neoplasms are represented mainly by MPN, MDS and AML [5]. According to the most recent data, HM are estimated to represent about 6.5 % of all cancers worldwide in 2012 [12].In the same year, NHL represented 2.7 % of all cancers and 2.4 % of all deaths from cancer worldwide.Leukemia accounted for 2.5 % of all cancers and 3.2 % of all deaths.MM represented 0.8 % of all cancers and 1.0 % of cancer deaths, while HL represented 0.5 % of all cancers and 0.5 % of cancer deaths [12].The incidence of HM varies from one country to another.It varies with geography, age and ethnicity, suggesting the involvement of different etiological factors for these diseases. In Morocco, there is no national database on hematological cancers, with the exception of some basic data found in the population-based cancer registries of Rabat and Casablanca [13][14][15].In Eastern Morocco, there is no population-based cancer registry or published epidemiology report on HM.Therefore, in this study, we present for the first time the epidemiological aspects of HM in Eastern Morocco.Analysis of their distribution and their relative frequency compared to all cancers in this region is also conducted. ", "section_name": "Background", "section_num": null }, { "section_content": "Eastern Morocco is located in the north east of the Kingdom of Morocco, and is the third largest region of the Kingdom.According to the High Commission for Planning (HCP), Eastern Morocco had a population of over 2 million in 2013.The population is mainly urban (67 % vs. 33 % rural) and young, nearly 6 out of 10 people are under 30 years [16].Our retrospective study was carried out in three centres: (1) Al-Farabi Regional Hospital (ARH) with its hematology and internal medicine units, which typically manage HM cases.(2) The Boussif Diagnostic Center (BDC), which also diagnoses HM cases.(3) Hassan II Regional Oncology Center (ROC), which normally manages all solid cancer cases.The study population consists of all patients diagnosed with cancer at the participating centres between January 2008 and December 2012.Among these, all cases of HM have been retrieved and reviewed carefully.For these cases, the data collected included details such as name, gender, age, place of residence, date of diagnosis, and type of HM.We have encountered some practical difficulties related to data collection since there is no methodic and effective system for data recording at the participating centres.For example, the same patient happens to be registered at the same center with slightly different names.So, before starting the analysis of the actual data, we proceeded to the elimination of duplicate cases.This step was necessary to avoid overestimating the actual case of diagnosed cancers. The FAB (French-American-British) classification system has been taken into account in the analysis of myeloid and lymphoid neoplasms [17][18][19].This classification was used instead of the 2008 WHO classification because of the lack of immunophenotypic, cytogenetic and molecular data necessary for the 2008 WHO classification.Data collection was performed on Excel.Statistical analysis was performed using SPSS software version 21.0.For the Chisquared test, the results are considered significant when p (degree of significance) is less than 0.05, very significant when p < 0.01 and highly significant when p < 0.001. In this retrospective study, obtaining informed consent was not possible.So, we were granted a waiver of consent by the Ethical Review Committee, and patient records/information was anonymized and de-identified prior to analysis.The study was approved by the Ethic Committee of the Faculty of Medicine and Pharmacy of Casablanca under the number 41/14.The authorization for personal data processing was obtained from the National Commission of control of Personal Data Protection under the number A-RS-280/2014. ", "section_name": "Methods", "section_num": null }, { "section_content": "In this study, 660 cases of hematological neoplasms were registered between January 2008 and December 2012 in the three participating centres.Overall, 6075 cases of cancers all sites combined were registered in these centres, indicating that HM account for around 10.9 % (660/6075) of all cancers recorded (Table 1).Lymphoid neoplasms were more common with 8.4 % of all cancers, and myeloid neoplasms accounted for 2.2 % of all cancers (Table 1 1).The median age difference observed between the various types of HM was statistically significant (p < 0.05).HM all types combined were slightly more common in men than in women with a male to female ratio of 1.1 (Table 1).This slight male predominance was statistically significant (p < 0.05).In males, NHL was the most frequent HM with 32 % of all male hematological cancers, followed respectively by HL (14.4 %), MM (12.7 %), MPN (9.8 %), CLL (8.1 %), AML (4.3 %), ALL (2.6 %), and finally MDS (2.3 %) (Table 1).In females, NHL was also the most frequent HM with 27.2 % of all female hematological cancers followed respectively by HL (17.6 %), MPN (16 %), MM (12.1 %), AML (5.4 %), MDS (3.8 %), CLL (3.2 %), and finally ALL (2.2 %).Only 4 cases of WM were registered in our study and were all female cases (Table 1). In our study, the overall median age at diagnosis for all HM combined was 54 years (Table 1).As with most cancers, the probability of being diagnosed with a HM increases markedly with age.However, unlike many other cancers, HM can be diagnosed at any age.Among the 660 registered cases of HM, the age at diagnosis was available for 651 cases.Of these, 51 cases were diagnosed in patients under 20 years of age, 119 cases in patients aged 20-39 years, 218 cases aged 40-59 years, and 263 cases were observed in patients aged 60 and over.The distribution of HM varies according to age group, and this difference was statistically significant (p < 0.05). Indeed, in patients under 20 years of age, HL was the most common with 39.2 % of HM diagnosed in this age group, followed respectively by NHL (29.4 %), AML (5.9 %), MPN (3.9 %); and ALL, MDS and MM (equally represented with 2 %) (Fig. 1, Additional file 1).In this age group, there is a male predominance with a male to female ratio of 1.8 (Fig. 1). In young adults aged 20-39 years, HL was also the most frequent with 31.9 % of HM diagnosed in this age group, followed respectively by NHL (18.5 %), MPN (13.5 %), AML (8.4 %), ALL and MM (equally represented with 4.2 %), CLL (1.7 %), MDS and WM (equally represented with 0.8 %) (Fig. 1, Additional file 1).In this age group, there is a slight male predominance with a male to female ratio of 1.2 (Fig. 1). In adults aged 40-59 years, NHL was predominant with 36.2 % of HM diagnosed in this age group, followed and WM (0.9 %) (Fig. 1, Additional file 1).In this age group, there is a male predominance with a male to female ratio of 1.3 (Fig. 1). In older individuals over 60 years old, NHL was the most common with 29.3 % of HM, followed respectively by MM (19.0 %), MPN (11.0 %), CLL (9.5 %), HL (9.1 %), MDS (4.6 %), AML (3.4 %), ALL (2.3 %) and WM (0.4 %) (Fig. 1, Additional file 1).In this age group, there is a female predominance with male to female ratio of 0.9 (Fig. 1).The sex ratio difference between the various age groups was statistically significant at (p < 0.05). In the group of lymphoid neoplasms, NHL was the most common disease with 38.7 % of all lymphoid neoplasms, followed respectively by HL (20.7 %), MM (16.2 %), CLL (7.5 %), ALL (3.2 %), and WM (0.8 %) (Fig. 2a).Among the 196 NHL cases recorded during the study period, only 46 cases had information on the histological type available.The analysis of these showed that the diffuse large cell lymphomas are the most common (45.7 %), followed respectively by diffuse small cell lymphomas (15.2 %), follicular lymphoma (8.7 %) and cutaneous lymphoma (4.3 %) (Fig. 3).Analysis of ALL showed that L2 was the predominant FAB subtype with 67 % of ALL, followed respectively by L3 (22 %) and L1 (11 %) (Fig. 4a).For myeloid neoplasms, MPN were the most frequent with 61.8 % of all myeloid neoplasms, followed by AML (23.5 %) and MDS (14.7 %) (Fig. 2b).Among FAB subtypes of AML, the commonest was M2 (46 %) followed by M3 and M5 (17 % each), M1 (8 %), and M0, M4 and M6 (4 % each) (Fig. 4b). ", "section_name": "Results", "section_num": null }, { "section_content": "To our knowledge, this is the first epidemiological study on HM in Eastern Morocco.It was carried out using data collected from three different tertiary health care facilities available for management of cancer patients.It presents for the first time the relative frequency of several types of HM compared to all cancers in this region of Morocco.In this study, HM constituted 10.9 % of all cancers.This result is similar to those of Rabat and Casablanca [13,15,20].In The United-States, HM accounted for 9,3 % of all cancers [21], which is similar to our data, but a much higher percentage was observed for hematological cancers in other studies: (18.05 % in Nigeria, 20 % in Iran and 24.8 % in Yemen) [22][23][24][25].We found that lymphoid neoplasms are the most common, accounting for 76.8 % of all HM.This result is similar to those observed in cancer registries of Rabat, and Casablanca [13,15].Lymphoid neoplasms are also predominant in North America, Australia, Europe, Africa and Asia [26][27][28][29][30]. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Sex-ratio of HM We found that overall men are slightly more affected with HM than women, with a male to female ratio of 1.1 (p < 0.05).Such a finding is similar to those reported in France and in the United Kingdom, where male to female ratio was 1.2 and 1.3, respectively [28,30].However, the sex ratio was much higher in Bangladesh and Senegal with a male to female ratio of 2.2 and 1.6, respectively [29,31].In the population of Rabat, males were also much more affected than females with HM [15,20]. Although there is a slight male predominance among HM overall, the sex ratio varied between age groups.In our study, a strong male predominance was observed in the age group 0-19 years, with a male to female ratio of 1.8.A similar result was observed in the Casablanca registry for this age group, with a male to female ratio of 2.5 [13].Several studies have shown that in developing countries HM in children often affect boys more than girls [32].For the age groups 20-39, 40-59 and 60 years and over, the female to male ratio was 1.2, 1.3 and 0.9, respectively.This underlines the importance of the sex variable in the development of HM, as has been noted previously [33].Further research is needed to understand the sex differences observed in Eastern Morocco. ", "section_name": "Sex-ratio and age of HM in Eastern Morocco", "section_num": null }, { "section_content": "Our study revealed that HM in Eastern Morocco occur in adult population with an overall median age at diagnosis of 54 years (mean age 51.96 years).In Western countries, the HM usually affect older people.For example, in the United Kingdom, the median age at diagnosis was 70.6 years [28].However, in Asia, HM usually affect the younger population.For example, in Bangladesh, the median age was 42 years [31].Thus, the median age at diagnosis of HM in Eastern Morocco is younger than that found in Western countries, but it is higher than that in Asia.It is important to note that the real median age in Eastern Morocco could be even ", "section_name": "Median age at diagnosis of HM", "section_num": null }, { "section_content": "In our study, the majority of hematological cancer cases have been observed among patients aged 60 years and over (40.4 % of MH).This result is predictable since cancer is triggered after several genetic mutations that accumulate with age [34,35].In this age group, there was a female predominance with a male to female ratio of 0.9.The greater longevity of women might be partly responsible for this female predominance among elderly patients.In this age group, NHL was the most common (29.3 % of HM), followed respectively by MM (19.0 %), MPN (11.0 %), CLL (9.5 %), HL (9.1 %), MDS (4.6 %), AML (3.4 %), ALL (2.3 %) and WM (0.4 %).The higher prevalence of HM in this age group justifies the need to develop effective programs to improve the management of hematological malignant diseases in the elderly, especially when they are associated with comorbidity factors [36,37]. ", "section_name": "HM in patients aged over 60 years", "section_num": null }, { "section_content": "In adolescents (15-19 years age group) lymphomas were predominant and accounting for 64.4 % of HM (HL-33.3%, NHL-31.1 %).Lymphomas were also the most common cancers among adolescents in Europe, North America, Middle East, Algeria and several African countries [38][39][40][41][42].However, in Asia and Latin America, leukemias were the most frequent cancers in adolescents [31,42,43].The distribution of lymphomas in our study is comparable to that in Europe, North America and Israel where HL is more frequent than NHL [38,42].However, in Algeria and in most African countries, HL is less common than NHL [38][39][40][41][42]. Infection with Epstein-Barr virus (EBV) has been associated with most cases of NHL or HL [44][45][46][47].Other risk factors have been associated with HM in children such as diet, paternal smoking, genetic and environmental factors, but the results are inconclusive [48][49][50].Further studies are needed to understand the reasons for this distribution of HM in adolescents in Eastern Morocco. ", "section_name": "HM in adolescents", "section_num": null }, { "section_content": "", "section_name": "Features of lymphoid neoplasms in Eastern Morocco", "section_num": null }, { "section_content": "In this study, NHL was the most frequent common HM.NHL was also the most frequent HM in cancer registries of Rabat, Casablanca and other African countries [13,15,20,23,51,52].In developed countries, NHL is also the most prevalent type of HM, with the highest incidence in the United States, Australia, New Zealand and Europe [53].In Asian countries like Japan, Korea and Yemen, NHL is also the commonest cancer among HM.However, in other countries including India and Bangladesh, leukemia cases were the most frequent HM [12,31,43,53,54]. NHL was more common in men than in women with a male to female ratio of 1.3.Such a finding is similar to that reported in Casablanca, where male to female ratio was 1.53 [13,55].However, the sex ratio was much higher in Rabat with a male to female ratio of 2.25 [20].The frequency of NHL increases with advancing age, but affected equally adults and elderly (Fig. 5).We observed that 7.8 % of all NHL were under 20 years, 37.9 % were below age 50 and 39.9 % were over 60 years (Fig. 5, Additional file 1).This age distribution of NHL is similar to that observed in Casablanca where patients under 20 accounted for 8 % of all NHL cases, patients younger than 50 years accounted for 38 %, while those over 60 years accounted for 42.1 % [13,55].In our study we found that diffuse large cell lymphoma was the most common form of NHL (45.7 %), which is consistent with the Rabat and Casablanca studies, which also reported a predominance of diffuse large cell lymphoma with 46.9 % and 50.1 %, respectively [20,55].Diffuse small cell lymphoma constituted 15.2 % of NHL, which is lower compared to Casablanca (19.2 %) [55].Follicular lymphoma constituted 8.7 %, which is higher than the figure suggested by the Rabat study (6.2 %) [20].Cutaneous lymphoma constituted 4.3 % of NHL, which is comparable to that in Casablanca (3.3 %), and Rabat (6.2 %) [20,55].Further extensive study is necessary to understand this difference in the distribution of the various form of NHL between Eastern Morocco and other regions of Morocco. ", "section_name": "Non-Hodgkin's lymphoma", "section_num": null }, { "section_content": "In this study, HL was the second most common blood cancer after NHL, accounting for 15.9 % of all HM.This frequency is lower than that observed in the cancer registry of Casablanca (between 17 % and 21.4 %), but it is higher than those observed in Rabat (10.4 %), United States (5.87 %), and France (5 %) [20,21,30,55,56].A lower frequency was observed in other studies in Asian countries including China, Japan, India, Korea and Bangladesh [31,[57][58][59][60].In our study, HL occurred in people of all ages with a peak in young adults (36.9 % of all patients with LH occurred in the age group 20-39 years) (Fig. 5, Additional file 1).In developed countries, the age distribution of HL is bimodal, the first peak occurs in young adults (20-34 years) and the second peak in the older age group (55-74 years).In contrast, in developing countries, the first peak occurs in children (under 20), probably because of an earlier infection with the Epstein-Barr in children living in those countries [61,62].Therefore, the age distribution of HL in Eastern Morocco resembles that of developed countries with a peak in young adults. In our study, we found that women are slightly more affected with HL than men, with a male to female ratio of 0.9.Such a finding is far from that reported in other studies, where HL is much more frequent in men than in women with a male to female ratio between 1.5 and 2 [33,63,64].In Casablanca and Rabat, it was reported that HL affects males and females, with an equal ratio [13,20].Additional studies are needed to better understand these sex ratio differences. ", "section_name": "Hodgkin's lymphoma", "section_num": null }, { "section_content": "MM is the fourth most common blood cancer in Eastern Morocco, accounting for 12.4 % of all HM.This frequency of MM is comparable to those in the United States (13 %), France (13.7 %) and UK (10.5 %) [28,30,65].We found that men are slightly more affected with MM than women, with a male to female ratio of 1.2, which is consistent with other studies [33,65,66].The frequency of MM increases with advancing age: 1.2 % MM occurred in children under 20 years, 6.1 % in young adults, 31.7 % in adults and 61 % in patients aged 60 years and over (Fig. 5, Additional file 1).The overall median age at diagnosis was 63.5 years, which is significantly higher as compared to Asian countries where the median age is around 55 years [31,67], but it is lower as compared to Western countries, where the median age is between 65 and 70 years [68]. ", "section_name": "Multiple Myeloma", "section_num": null }, { "section_content": "In this study, CLL constituted 5.8 % of all HM.This frequency is higher as compared to those observed in Asian countries [31,69,70], and lower as compared to what has been reported in Western countries [27,28,30,71].In the US, the incidence of CLL is lower among Asians and African-Americans as compared to Caucasians [72].Therefore, multiple factors may play an important role in the development of CLL, including genetic and environmental factors [71][72][73]. We found that the median age at diagnosis for CLL was 67 years.This result is intermediate between the results found in Western countries (median age of 70-72 years) and Asia (median age of 59-60 years) [28,30,31,70,74].We found that the frequency of CLL increases with advancing age.Indeed, no cases of CLL have been observed in children under 20 years, 5.3 % of CLL occurred in young adults, 28.9 % in adults and 65.8 % in patients aged 60 years and over (Fig. 5 ", "section_name": "Chronic lymphoid leukemia", "section_num": null }, { "section_content": "There is a male predominance among CLL, with a male to female ratio of 2.8, which is in accordance with studies reported from India (sex ratio of 3), Bangladesh (sex ratio of 2.9), Ethiopia (sex ratio of 3.6), and Western countries (sex ratio of 1.5-2) [30,31,70,71,75]. ", "section_name": ", Additional file 1).", "section_num": null }, { "section_content": "ALL constituted 2.4 % of all HM.This frequency is similar to those reported from France (2.7 %), UK (2 %) and United States (3.84 %) [21,28,30], but lower as compared to those observed in Asian countries such as Yemen (4 %) and Bangladesh (14.1 %) [22,31].ALL is generally most common in childhood and its age incidence curve is bimodal, with a peak in childhood and another peak in old age [30,63,76].Our study showed a different profile, with the first peak occurring in the 20-39 years age group, and the second peak in old age (Fig. 5, Additional file 1).Only 6.3 % of ALL was observed in children aged under 20 years old, and thus the childhood peak was absent.This difference could be explained by the under-representation of children in our study, since children HM are usually treated in other services.We also found that L2 was the predominant FAB subtype (67 %), followed by L3 (22 %) and L1 (11 %), which is consistent with the age distribution of ALL in our study since L2 is more often observed in adults and most childhood cases are of subtype L1 [77]. ", "section_name": "Acute lymphocytic leukemia", "section_num": null }, { "section_content": "", "section_name": "Features of myeloid neoplasms in Eastern Morocco", "section_num": null }, { "section_content": "In our study, MPN constituted 12.7 % of all HM.This frequency is comparable to that reported in France (11 %), and the UK (9 %) [28,30].We found that MPN were more common in women than in men, with a male to female ratio of 0.7.A female predominance was also observed in the United Kingdom (sex ratio of 0.8) and France (sex ratio of 0.94) [28,30].We also found that MPN were more common in adults and elderly but also affected younger patients (Fig. 5, Additional file 1).The mean age of diagnosis was 52.8 years, which is lower than the one observed in France (65) [30].The younger age phenomenon of HM was discussed earlier in this discussion. ", "section_name": "Myeloproliferative neoplasms", "section_num": null }, { "section_content": "AML constituted 4.9 % of all HM.This frequency is comparable to that in Yemen (4 %), but it is lower than those reported in France (7 %), UK (6.68 %), United States (12 %) and Bangladesh (28.3 %) [21,22,28,30,31].We found that AML is more common in women than in men with a male to female ratio of 0.9 which is different from what was found in other studies [28,30,31,76,78].Our study showed that AML affected equally young adults, adults and elderly (Fig. 5, Additional file 1).However, in Western countries, AML generally affects older people with a median age of 65-67 years [71,79].In France, for example, 60.6 % of AML was observed in people aged 60 years and over [30].A different picture is observed in Asia, where AML affects young adults with a median age at diagnosis of 30 years in India and 35 in Bangladesh [31,80].In our study, the distribution of AML according to the FAB classification, has revealed that M2 was the predominant FAB subtype (46 %), followed by M3 and M5 (17 %), M1 (8 %), M0, M4 and M6 (4 %).This distribution was comparable to that reported in China [81].The predominance of M2 was also observed in Iran, India, Germany, USA and Singapore [82][83][84][85][86]. ", "section_name": "Acute myeloid leukemia", "section_num": null }, { "section_content": "MDS constituted 3 % of all HM, which is lower to what was reported in Bangladesh (4.5 %), UK (6 %), and France (12.8 %) [28,30,31].We found that MDS affected elderly with a median age of 62 years, and 60 % of the MDS patients aged over 60 years (Fig. 5, Additional file 1).This result is similar to that reported in Western countries where MDS typically affects elderly people with a median age at diagnosis between 60 and 70 years [28,30].However, in Asian countries, MDS typically affects people who are much younger [31,43,80].The median age was 46.1 years in India and 57 years in Bangladesh [31,87].We found that MDS is more common in women than in men with a male to female ratio of 0.7, which is different from what was found in other studies.For example, the sex ratio was 1.38 in France, 2 in UK, and 1.9 in Bangladesh [28,30,31]. ", "section_name": "Myelodysplastic syndrome", "section_num": null }, { "section_content": "This study provided for the first time the pattern and distribution of HM in Eastern Morocco.HM occur at a relatively young age, with an overall median age at diagnosis of 54 years.Overall, men are slightly more affected with HM than women, with a male to female ratio of 1.1.NHL was the most common HM accounting for 29.7 % of all HM, followed by HL, NMP, MM, CLL, AML, MDS, ALL, and WM.The majority of HM cases have been observed among patients aged 60 years and over (40.4 % of MH).In adolescents, HL was the most frequent HM. Our findings justify the need to establish a regional cancer registry as a first step in blood cancer control in Eastern Morocco.Further studies are necessary to better understand and develop a more effective program aiming at controlling and preventing the spread of blood cancers in Eastern Morocco. for their support.We also thank the Regional Director of the Ministry of Health in Eastern Morocco for his support.We gratefully acknowledge Dr. N. Abda's help with the data analysis.We thank Dr A. Azzouzi and all the administrative staff of the Faculty of Medicine and Pharmacy of Oujda for their valuable support and encouragement throughout the entire work.The ", "section_name": "Conclusion", "section_num": null } ]	[ { "section_content": "No funding was received for this study.We are grateful to the clinical team and to all administrative staff at the participating centres (Al-Farabi Regional Hospital, Boussif Diagnostic Center, and Hassan II Regional Oncology Center of Oujda), for allowing their health facilities to participate in the study and authors thank Dr. Amar Sellam for revising the English version of the manuscript. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "Additional file 1: Includes two tables: (Table S1) showing the distribution and male to female ratio of hematological malignancies by age group in Eastern Morocco for the study period, and (Table S2) showing the Age-group specific distribution of hematological malignancies in Eastern Morocco, 2008-2012.(PDF 32 kb) Abbreviations ALL: Acute lymphoblastic leukemia; AML: Acute myeloid leukemia; CLL: Chronic lymphocytic leukemia; HL: Hodgkin's lymphoma; HM: Hematological malignancies or Hematological malignancy; MDS: Myelodysplastic syndromes; MM: Multiple myeloma; MPN: myeloprofilerative neoplasms; NHL: Non-Hodgkin's lymphoma; WM: Waldenström macroglobulinemia. The authors declare that they have no competing interests. Authors' contributions MEE, MEE, RB and MB conceived and designed the study.MEE, MEE, and MB, carried out the analysis.MEE and MB drafted the initial manuscript.MEE, MEE, RB, MO and MB participated in interpreting the data and in revising the manuscript.All authors read and approved the final manuscript. ", "section_name": "Additional file", "section_num": null }, { "section_content": "Additional file 1: Includes two tables: (Table S1) showing the distribution and male to female ratio of hematological malignancies by age group in Eastern Morocco for the study period, and (Table S2) showing the Age-group specific distribution of hematological malignancies in Eastern Morocco, 2008-2012.(PDF 32 kb) Abbreviations ALL: Acute lymphoblastic leukemia; AML: Acute myeloid leukemia; CLL: Chronic lymphocytic leukemia; HL: Hodgkin's lymphoma; HM: Hematological malignancies or Hematological malignancy; MDS: Myelodysplastic syndromes; MM: Multiple myeloma; MPN: myeloprofilerative neoplasms; NHL: Non-Hodgkin's lymphoma; WM: Waldenström macroglobulinemia. ", "section_name": "Additional file", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. Authors' contributions MEE, MEE, RB and MB conceived and designed the study.MEE, MEE, and MB, carried out the analysis.MEE and MB drafted the initial manuscript.MEE, MEE, RB, MO and MB participated in interpreting the data and in revising the manuscript.All authors read and approved the final manuscript. ", "section_name": "Competing interests", "section_num": null } ]
10.7150/ijbs.83302	Loss of MIR503HG facilitates papillary renal cell carcinoma associated lymphatic metastasis by triggering NOTCH1/VEGFC signaling	Clinical lymphatic metastasis indicates an extremely poor prognosis. Patients with papillary renal cell carcinoma (pRCC) have a high probability of progressing to lymphatic metastasis. However, the molecular mechanism of pRCC-associated lymphatic metastasis has not been elucidated. In this study, we found a downregulated long non-coding RNA (lncRNA) MIR503HG in pRCC primary tumor tissues due to hypermethylation at the CpG islands within its transcriptional start site. Decreased MIR503HG expression could stimulate tube formation and migration of human lymphatic endothelial cell (HLEC) and play a central role to promote lymphatic metastasis in vivo by enhancing tumor lymphangiogenesis. MIR503HG, located in the nucleus, bound with histone variant H2A.Z and affected the recruitment of histone variant H2A.Z to chromatin. Subsequently, increasing the H3K27 trimethylation caused by MIR503HG-overexpression epigenetically downregulated the NOTCH1 expression, which ultimately resulted in decreasing VEGFC secretion and lymphangiogenesis. Additionally, downregulated MIR503HG facilitated the HNRNPC expression, which ultimately promoted the maturation of NOTCH1 mRNA. Notably, upregulating MIR503HG expression might decrease pRCC resistance to the mTOR inhibitor. Together, these findings highlighted a VEGFC-independent mechanism of MIR503HG-mediated lymphatic metastasis. MIR503HG, identified as a novel pRCC-suppressor, would serve as the potentially biomarker for lymphatic metastasis.	[ { "section_content": "Papillary renal cell carcinoma (pRCC), which varies widely from other RCC histologic variants, has its genetic and pathologic characteristics [1].Ranked as the second-most type of RCC, it represents 10-15% of all kidney cancers [2,3].Different clinical behaviors were discovered in RCC subtypes because of biological distinction [4,5].As the pattern of manifestation of biological underpinnings, pRCC has greater tendency to become metastatic pRCC (mpRCC) and spreads in different sites in patients. Compared with other subtypes, lymph node (LN) is recognized as the most common metastatic site in pRCC [6].Up to now, this unique biologic profile of pRCC has not been elucidated.Through the lymphatic system, cancer cells could be disseminated to lymph-vessels and transported to distant LNs, finally settled down and colonized in LN [7].Therefore, lymphangiogenesis, as an essential process for lymphatic metastasis, was associated with metastasis-free survival in a dozen Ivyspring International Publisher solid tumors [8,9]. Previous studies reported the molecular markers can distinguish lymphatic vessels from blood vessels.Vascular endothelial growth factor C, named VEGFC, could induce lymphangiogenesis in tumor tissues [10].Given that the VEGFC/VEGF receptor 3 signaling was the key pathway for lymphatic metastasis [11,12], multiple targeted drugs (anti-lymphangiogenic) have undergone clinical trials, such as monoclonal antibodies VGX-100 and IMC-3C5 [13,14].Understanding the specific mechanisms of VEGFC signaling pathway in pRCC would provide valuable treatment options for effective anti-VEGFC treatment in the clinic. Long non-coding RNA (lncRNA) is a type of transcripts that are longer than two hundred nucleo-tides [15].Although exhibiting no protein-coding potential, it was reported that it involved in many biological processes, including regulating chromatin looping and modification [16], functioning as enhancer-associated RNAs [17,18], trapping for some transcription factors [19], etc.Recent years, several researches have gradually unveiled the pathophysiological contributions of lncRNAs in the lymphatic metastasis [20][21][22].Untill now, few studies have explored the function and mechanism of lncRNAs of pRCC-associated lymphatic metastasis. In the present study, we discovered MIR503HG, a downregulated lncRNA in pRCC, exerted a pivotal role in the VEGFC axis.Our finding revealed a novel mechanism of MIR503HG-related lymphatic metastasis for pRCC.Representative image of Western blotting analysis of VEGFC protein expression in MIR503HG depletion (g) and overexpression (h) in pRCC cells.i-j.Representative images of the wound-healing assay using pRCC cell lines showing cell motility after ectopic expression (j) or knockdown of MIR503HG (i) at indicated time are shown.k.Invasion assays of pRCC cells with MIR503HG knockdown and overexpression.In all panels, data are shown as the mean ± s.d.For all representative images, at least three independent experiments were performed with similar results.NS, not significant, P < 0.05, P < 0.01, *P < 0.001, one-way ANOVA followed by Dunnett's post hoc test was performed in e-f.The two-tailed Student's t-test was used in i-k, and the nonparametric Mann-Whitney U test was used in b-d.Table 1.Downregulated lncRNAs according to the GEPIA dataset. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Using transcriptome data from Gene Expression Profiling Interactive Analysis (GEPIA) database, we determined lymphatic metastasis-related lncRNAs in pRCC (Fig. 1a).In short, we firstly selected 48 eligible lncRNAs (Table 1) and then 8 lncRNAs were identified as the pRCC-associated lncRNAs, all of them were only downregulated in kidney renal papillary carcinoma (KIRP), but not in kidney renal clear cell carcinoma (KIRC) or kidney chromophobe (KICH).Top 3 downregulated lncRNAs were selected as candidates to explore the crucial lncRNA involved in pRCC-associated lymphatic metastasis (Fig. 1b-d). We respectively established lncRNA-knockdown pRCC cell lines: CAKI-2 and ACHN.These two cell lines were widely reported the harbor genomic characteristics of pRCC [25,26].Next, with the ELISA assays, we found that MIR503HG was negatively associated with the VEGFC secretion in both CAKI-2 and ACHN cells (Fig. 1e-f), which was then verified by western blotting assay (Fig. 1g-h).Shifting the emphasis from three candidates to LncRNA-MIR503HG, we next established stable MIR503HG knockdown (shRNA) and overexpression pRCC cell lines for further research.The efficiency of siRNA, shRNA and overexpression were confirmed by qRT-PCR (Supplemental Fig. 1a-h).MIR503HG, as a tumor suppressor gene, could inhibit several solid tumors' capacity of progression [23,24].Our results showed that MIR503HG knockdown significantly enhanced invasion (Transwell assays) and migration (Wound-healing assays) of CAKI-2 and ACHN cells compared with the control group.Correspondingly, compared with negative control group, MIR503HG overexpression reduced not only migratory but also invasive capacity in both CAKI-2 and ACHN cells (Fig. 1i-k). ", "section_name": "The pRCC-associated lncRNA-MIR503HG negatively correlated with VEGFC secretion", "section_num": null }, { "section_content": "As expected, we found that MIR503HG expression was especially downregulated in several pRCC cell lines (UO31, ACHN and CAKI-2) in the Cancer Cell Line Encyclopedia (CCLE) datasets (Fig. 2a).Based on the qRT-PCR results of Renji pRCC-cohort, we observed that lower expression of MIR503HG was associated with high rate of lymphatic metastasis (Fig. 2b), high tumor grade (Fig. 2c) and poor PFS (Fig. 2d).Using the pRCC tissues from patients, we established patient derived xenograft (PDX) models for determining its suppressive role in pRCC-associated lymphatic metastasis (Fig. 2e).Patient-KC67 with the lower MIR503HG expression was diagnosed as the lymphatic metastasis, which was examined by magnetic resonance imaging (MRI) during follow-up (Fig. 2f-g).Patient-KC67 also showed high microlymphatic vessel density, which was indicated by LYVE-1 positive region (Fig. 2h).Two patients' clinical information were collected (Table 2). ISH assays were also performed by using formalin fixed paraffin-embedded (FFPE) tissues obtained from pRCC patients.Statistical analysis revealed that MIR503HG expression was significantly associated with lymphatic metastasis, suggesting it might be a prognostic factor of pRCC (Fig. 2i).As expected, we noticed that MIR503HG could be detected in pRCC cells' nucleus in subcellular fractionation assay (Fig. 2j), which was consistent with results of ISH.Furthermore, tube formation experiments were performed in order to explore regulatory function of MIR503HG in lymphangiogenesis of pRCC.HLECstube formation was significantly inhibited after incubating with conditioned medium from MIR503HG overexpression pRCC cells (Supplemental Fig. 2a), while promoted in the siMIR503HG pRCC cells, compared with the control group, respectively (Fig. 2k). ", "section_name": "Downregulated MIR503HG related with poor prognosis and lymphatic metastasis of pRCC", "section_num": null }, { "section_content": "In consideration of DNA methylation is the dominating mechanism regulating lncRNA expression, bioinformatic data mining was conducted, and DNA hypermethylation status was observed at the transcription start site of MIR503HG in both MethPrimer and EMBOSS database (Fig. 3a-b).As the data from MEXPRESS database shown that methylation level was higher in primary pRCC than normal tissue (Fig. 3c), there may be a negative correlation between methylation status and MIR503HG expression. We proposed that the downregulation of MIR503HG in tumor tissues was caused by DNA hypermethylation, a mainly epigenetic mechanism for eukaryotic genomes.Furthermore, refer to MEXPRESS, we selected 13 CpGs at MIR503HG promoter (Fig. 3d) to explore the DNA methylation status (cg-IDs were presented in Table 3).By bisulfite analysis, the methylation level of 13 CpGs was measured in 10 pairs of pRCC tumors and adjacent normal tissues.The average methylation status of CpG sites was higher in pRCC tissue than that in paired normal renal tissue (Fig. 3e-g).The methylation level of 3 specific CpGs (cg0174250, cg07776419, cg04109661) was remarkably increased in pRCC samples, but no significant difference was found in other CpGs (Fig. 3h, Supplemental Fig. 3a-j).MSP-PCR assays also confirmed that MIR503HG was significantly hypermethylated in pRCC tissues than in paired normal tissue (Fig. 3i).Importantly, the expression of MIR503HG in pRCC cells were induced after treated with 5'azacytidine (Fig. 3j).These results supported the hypothesis that DNA hypermethylation played a crucial role in the decrease of MIR503HG in pRCC. ", "section_name": "MIR503HG was downregulated in pRCC due to hypermethylation", "section_num": null }, { "section_content": "We conducted RNA pull-down assays to determine MIR503HG interacting proteins in pRCC.Silver staining revealed a specific protein region between 10-20 kDa in the MIR503HG pull-down samples as compared with antisense group (Fig. 4a and Supplemental Fig. 4a).Mass spectrometry assay was conducted in order to find the potential interacting proteins (Top 5 differential proteins identified were listed in Table 4).Notably, we found the potential binding partner protein, histone variant H2A.Z (Supplemental Fig. 4b), which has a well-known capability of regulating structure of chromatin, could participate in various biological processes by substituting the conventional histone proteins at specific locations of chromatin [27,28].Notably, lncRNAs have also been reported to interact with H2A.Z in the regulation process of cancer cells [29]. To confirm the interaction and map the RNA-binding region of H2A.Z, western blotting was performed (Fig. 4b) and serial deletion assays were generated and confirmed that 647-775 nt sequences (5 ′-terminal of MIR503HG) was the essential part for the interaction with H2A.Z (Fig. 4c-e), and removal of fragment 5 (647-775 nt) abolished the MIR503HG -H2A.Z interaction (Fig. 4f-g).We further predicted the putative binding sites of RNA-binding protein by MEME website (Supplemental Fig. 4c).The results from RNA pull-down assay and RIP assay simultaneously proved the exact interactions between 647-775 nt and H2A.Z (Fig. 4h-i).FISH and IF assays also indicated MIR503HG and H2A.Z co-localized in the pRCC cells' nucleus (Fig. 4j), suggesting MIR503HG could bind with H2A.Z. ", "section_name": "MIR503HG directly bound with H2A.Z", "section_num": null }, { "section_content": "Emerging evidences revealed that non-coding RNAs were widely participated in the regulation of cellular signals in tumor progression [33,34].In view of such phenomenon, the alteration of key genes in some important pathways related with RCC progression were detected after silencing MIR503HG expression in pRCC cells (Fig. 5a and Supplemental Fig. 5a-d).Notably, four pathways (Hedgehog, NOTCH/Jagged, WNT/β-catenin and TGF-β) with altered genes expression were further analyzed by ELISA assays.Rather than other pathway inhibitors, IMR-1, which specifically inhibited transcriptional activation complex on NOTCH chromatin [40], could reverse the stimulation by MIR503HG knockdown to VEGFC secretion (Fig. 5b and Supplemental Fig. 5e).Furthermore, consistent with the results of qRT-PCR, protein level of NOTCH1 expression were markedly increased by silencing MIR503HG expression.Conversely, levels of NOTCH1 were decreased in MIR503HG-overexpression pRCC cells (Fig. 5c).It is well-known that activation of NOTCH1 would promote RCC progression and metastasis [35,36].Kaplan-Meier curves also revealed that the high level of NOTCH1 expression was associated with poor overall survival in pRCC (Supplemental Fig. 5f).Additionally, NOTCH1-related signaling pathway could interact and regulate the expression level of VEGF family [37][38][39].According to all above, we came to a conclusion that NOTCH1 was indispensable for MIR503HG-mediated VEGFC secretion in pRCCassociated lymphatic metastasis. Given that H2A.Z was recognized as a crucial role in epigenetic modification of histone [30,31], indeed, it epigenetically modulated target genes expression by catalyzing H3K27 trimethylation (H3K27me3) [32].We transfected pRCC cells with corresponding small interfering RNA to prove that H2A.Z knockdown in pRCC cells rescued the inhibitory effect of MIR503HG overexpression on cell viability (Supplemental Fig. 5g) and silencing H2A.Z observably decreased H3K27 trimethylation level (Fig. 5d).Out of curiosity, we began to consider whether MIR503HG could regulate expression of NOTCH1 via H2A.Z-mediated H3K27 trimethylation.ChIP assays were designed and performed with anti-H2A.Z antibody and anti-H3K27me3 antibody, showing that H2A.Z was markedly enriched between -224 and +54 nucleotides (P1) in the promoter of NOTCH1 (Fig. 5e), MIR503HG-overexpression may dramatically increase H3K27me3 via coordinating depositions of H2A.Z in NOTCH1 promoter region (Fig. 5f and Supplemental Fig. 5h).Furthermore, luciferase report assays demonstrated that MIR503HG knockdown induced the luciferase activity of WT promoter of NOTCH1, while MIR503HG overexpression markedly decreased the luciferase activity (Fig. 5g and Supplemental Fig. 5i). ", "section_name": "Downregulated MIR503HG epigenetically upregulated NOTCH1 expression and promoted VEGFC secretion in pRCC", "section_num": null }, { "section_content": "To further elucidate whether MIR503HG could suppress the lymphatic metastasis by downregulating VEGFC, we developed orthotopic xenograft model by injecting cells into the subrenal capsule to determine whether antagonism of VEGFC could suppress shMIR503HG-stimulated lymphatic metastasis. IVIS showed that anti-VEGFC could reduce the shMIR503HG-induced metastatic capability of pRCC to LNs, and all suspicious tissues were collected and taken for histopathological observation to verify the lymphatic metastases (Fig. 6a-c).Consistently, IHC from LYVE-1 indicated that shMIR503HG significantly increased density of micro-lymphatic vessels in left-kidney capsule while treating with VEGFC neutralizing antibody markedly weakened shMIR503HG-stimulated lymphangiogenesis (Fig. 6d).On the other hand, antagonism of VEGFC significantly suppressed shMIR503HG-stimulated tube formation and migration of HLECs (Fig. 6e).Taken together, these results demonstrated that MIR503HG suppressed lymphatic metastasis in pRCC by regulating VEGFC. ", "section_name": "MIR503HG suppressed lymphatic metastasis through altering VEGFC in pRCC", "section_num": null }, { "section_content": "With silver staining, another differential binding band between MIR503HG and antisense sequence were also noticed (Fig. 7a).Heterogeneous nuclear ribonucleoprotein C (HNRNPC), as known as N6-methyladenosine (m6A)-associated protein, was identified (Table 4 and Supplemental Fig. 4d) and its interaction with MIR503HG was further validated by western blotting (Fig. 7b). It surprised us that there was a negative correlation between MIR503HG and HNRNPC (Fig. 7c).To illustrate the possibility that MIR503HG could regulate this m6A reader to influence its downstream target NOTCH1, we successfully established HNRNPC-KO cells through CRISPR/Cas9 approach (Supplemental Fig. 6a-c).Positive regulatory effects of HNRNPC on NOTCH1 expression were shown by western blotting assay (Fig. 7d-e).d.Expression level of NOTCH1 protein were measured by Western blotting analysis in pLenti-control and pLenti-sgHNRNPC.e.Expression level of NOTCH1 mRNA were measured by qRT-PCR analysis in pLenti-control and pLenti-sgHNRNPC.f.The potential m6A modification sites of NOTCH1 were predicted by SRAMP.g.RIP-qRT-PCR showing MIR503HG overexpression impaired the enrichment of m6A modification in the region P2.h.The enrichment of m6A were analyzed in NOTCH1-WT and NOTCH1-P2-MUT in the control and HNRNPC KO cells.i. RIP-qRT-PCR: detecting the enrichment of HNRNPC in MIR503HG KD in the NOTCH1-P2-MUT group.j.Pre-mRNA and mature NOTCH1 mRNA were analyzed using qRT-PCR after HNRNPC KO for 48 h.k.The survival curves of high and low HNRNPC expression were plotted by the Kaplan-Meier analysis with two-tailed log-rank test.Date was from The Cancer Genome Atlas (TCGA)-KIRP dataset.The statistical difference was assessed through 2-tailed Student's t-test in e, g-j.Error bars show the standard deviation (SD) from three independent experiments.P < 0.05; P < 0.01; P < 0.001. As known as the primary form of RNA modification, m6A regulates gene expression and decides eukaryote fate [41].To clarify whether NOTCH1 could be modified by m6A methylation, five m6A modification sites with very high confidence of NOTCH1 mRNA were predicted by SRAMP database (Fig. 7f and Supplemental Fig. 6d) and RIP-qPCR assays indicated that MIR503HGoverexpression significantly reduced HNRNPCbinding to the m6A-modified region (P2) of NOTCH1 (Fig. 7g-h and Supplemental Fig. 6e).Consistently, mutation of P2 abrogated the enrichment of HNRNPC in MIR503HG-silenced cells (Fig. 7i and Supplemental Fig. 6f). As widely reported, HNRNPC influenced the pre-mRNA processing, transport, stability, and other aspects of mRNA metabolism [42][43][44].Hence, we designed primers with or without introns, and performed qRT-PCR to determine that HNRNPC-KO decreased the efficiency of NOTCH1 mRNA processing and maturation (Fig. 7j).Moreover, similar to NOTCH1, Kaplan-Meier analysis of overall survival of TCGA-KIRP dataset revealed that higher expression of HNRNPC was associated with poor overall survival (Fig. 7k). Taken together, these findings indicated that downregulation of MIR503HG promoted maturation of NOTCH1 mRNA via HNRNPC mediating m6A-dependent manner. ", "section_name": "MIR503HG-modulated HNRNPC could maturate NOTCH1 mRNA in a m6A-dependent manner", "section_num": null }, { "section_content": "It was reported that LncRNAs enabled to predict the response to target therapy of RCC [45,46].In present study, to identify whether MIR503HG could be the potential therapeutic target of lymphatic metastasis, several drugs, such as Everolimus, Cabozantinib, Lenvatinib and Axinitib, which were currently used for RCC treatment, were examined during research.Western blotting assay showed that VEGFC and NOTCH1 expression were downregulated by treating several drugs, respectively (Fig. 8a and Supplemental Fig. 7a).Notably, qRT-PCR analysis revealed that MIR503HG expression level was significantly induced in Everolimus group, while no significant difference was found in other groups (Fig. 8b-d).Inhibition of mTOR signaling was reported that increases the expression of p53 protein level [47].Consistently, two potential p53-binding sites in MIR503HG promoter region were detected by JASPAR database (Supplemental Table 1).Interestingly, qRT-PCR analysis provided additional evidences for the mechanistic connection that Everolimus-enhanced MIR503HG expression was considerably decreased in cells with concurrent p53 silence (Supplemental Fig. 7b-c). It made us wonder that whether upregulating MIR503HG could enhance the sensitivity of pRCC cells to Everolimus treatment.Compared to control pRCC cells, shMIR503HG cells showed less sensitive to Everolimus (Fig. 8e).In addition, Everolimus treatment repressed NOTCH1 expression while depletion of MIR503HG minimized this effect (Fig. 8f).The pRCC cells were implanted into subrenal capsule of nude mice, consistent to survival analysis, metastasis sites assessed by IVIS confirmed that shMIR503HG reduced the therapeutic effectiveness of Everolimus in pRCC xenografts (Fig. 8g-h).Reducing MIR503HG expression in pRCC could partially reverse the negative effect of tumor-induced lymph-vessel formation caused by Everolimus (Fig. 8i).Collectively, our results suggested that MIR503HG might be a potential biomarker for pRCC treatment. ", "section_name": "Everolimus repressed lymphatic metastasis via inducing MIR503HG.", "section_num": null }, { "section_content": "MIR503HG, located on chromosome Xq26.3,differentially expressed in pRCC and exhibited values of prognosis.As several studies have previously focused on its function in cancer, it generally prevented tumor cells from proliferating, encroaching and migrating [48][49][50].In present study, we investigated LncRNA-MIR503HG, which was inversely correlated with cell invasion, migration and lymphangiogenesis, may have a close association with lymphatic metastasis of pRCC patients. Emerging evidences suggested that DNA methylation-mediated epigenetic alteration is one of the key processes controlling lncRNA expression and related-biological process.Moreover, this phenomenon exhibited a tissue specificity [51][52][53][54].By using bioinformatic data mining, MSP-PCR and Bisulfite sequencing, we confirmed the hypermethylation status in MIR503HG promoter.Subsequently, MIR503HG expression was proved to be negatively linked with methylation status.In our study, we observed that MIR503HG was involved in the cellular regulation by epigenetically inducing lymphangiogenesis and invasion of pRCC by increasing H3K27me3 via H2A.Z.Yuan et al. reported that lncRNAs may function by binding with H2A.Z in cancer cells [29], the interaction was proved to regulate the acetylation of H2A.Z.Our study proposed that MIR503HG might have an effect on the formation of H2A.Z-H3K27me3 complex with targeted DNA sequences.Dynamic regulation of H2A.Z in chromatin has been implicated in transcription process [55], it was reported to facilitate the recruitment of PRC2 for appropriate installation of H3K27 trimethylation at promoters of genes controlling cell development [56].However, due to the lack of detailed structure of H2A.Z protein itself, the mechanisms through which potential DNAbinding domains to target sites across the chromatin were still unclear. VEGFC, as a lymphatic vessel-specific growth factor, has been demonstrated that it played essential role in lymphatic metastasis, including compromising the endothelial lymphatic barrier, allowing for lymphatic invasion, and ultimately promoting lymphatic metastasis and causing treatment failure [57][58][59].Interdicting the VEGFC/VEGFR-3 axis has been widely reported to reduce the rate of lymphatic metastasis in a number of tumor-bearing experimental invivo models [60][61][62].Our results enhanced mechanistic understandings about pRCC-lymphatic metastasis, suggesting blockage of VEGFC reversed the effect of downregulated MIR503HG on lymphatic metastasis. It's well-acknowledged that disorders of m6A modification in RNA are closely related to the development of solid tumor [63].Jin et al. demonstrated that HNRNPC, as known as a m6A \"readers\", could mature DDX58 transcript by binding to m6A sites [64].It was also found that HNRNPC interacted with lncRNA CYTOR, and stabilized ZEB1 mRNAs by inhibiting nondegradative ubiquitination [65].However, its specific mechanism in pRCC has not been elucidated.We showed that downregulated MIR503HG could promote lymphatic metastasis by inducing HNRNPC.Moreover, HNRNPC was able to regulate downstream signal transduction through the N6-methylation of NOTCH1, which further promotes the secretion of VEGFC in pRCC cells.Our findings would provide a new perspective on the regulation of m6A modification in lymphatic metastasis. There is no consensus among clinicians regarding which drug was the optimal treatment option for pRCC-associated lymphatic metastasis.Indeed, previous research has reported that the activation of mTOR was a widespread event in clinical samples of invading locoregional LN [66].In prostate cancer, increasing p-mTOR expression was demonstrated that has positive correlations with lymphangiogenesis and lymphatic metastasis [67].Specifically, AKT/mTOR signaling pathway acted as a critical role of promoting lymphatic metastasis in cervical cancer and medullary thyroid carcinoma [68,69].However, further works remain to be done to define precisely how mTOR inhibitors acts in lymphatic metastasis of pRCC. TKIs and mTORs are most common therapeutic options among the several classes of available treatments.Everolimus, as a classic mTOR inhibitor, is recommended as a second-line treatment for metastatic RCC patients previously received at least one TKI.On the pRCC front, however, there is little research evidence of everolimus treatment. As a single-arm phase II trial with everolimus in previously untreated pRCC, the RAPTOR trial has generally received limited attention, this trial enrolled totally 88 patients in its intention-to-treat (ITT) cohort [70].Despite a very low response rate (1%), an OS of 21.4 months (95% CI, 15.4-28.4)were noteworthy.A large percentage of patients (65%) in this trial showed stable disease (SD) as a best response.As results of RAPTOR, mTOR inhibition will be a primary tactic in pRCC. The effectiveness of doublet therapy was then discussed in light of single-agent targeted therapy's success in non-ccRCC.Hence, more possible strategies need to carry on to countering resistance mechanisms.Hutson.et al. assessed the regimen in nccRCC based on the efficacy of lenvatinib with everolimus after one line of VEGF-directed therapy [71], which means combinations may improve the treatment effectiveness of pRCC. It's well known that ccRCC derived great benefit from VEGFR-associated multi-targeted TKIs, such as sunitinib, pazopanib, axitinib.According to our results, however, TKIs did not have any impact on MIR503HG expression level in PRCC.In our opinion, its mechanism may by realized not by directly regulating MIR503HG, but by other indirect signals.MIR503HG interacts with signaling pathways, such as inhibiting the phosphorylated proteins of RAF, MEK, ERK1/2, PI3K and AKT [72].In view of PI3K inhibitors were determined that could reverse TKIs resistance in tumor [73], and activated AKT promoting TKIs resistance in RCC [74], we speculate that MIR503HG-KO cells will be more sensitive to those TKIs than control cells. Based on our work, we confirmed that inducing MIR503HG may have therapeutic benefit for sensitizing Everolimus treatment on pRCC and its associated lymphatic metastasis.This finding might become a unique therapeutic approach for us to suppress pRCC. ", "section_name": "Discussion", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "67 RNA later-preserved pRCC specimens were collected from pRCC patients underwent radical nephrectomy at Renji Hospital (Shanghai, China) between June 2013 and July 2020.The clinicopathological characteristics were summarized in detail (Table 5).After surgery, clinical samples, including tumor and adjacent normal tissue were immediately snap-frozen in liquid nitrogen and stored in cryogenic refrigerator at -80 °C.Formalin-fixed and paraffin-embedded (FFPE) tumor tissues were obtained and histologically confirmed by three pathologists. ", "section_name": "Clinical data and specimens", "section_num": null }, { "section_content": "Cell lines involved in this study were all purchased from American Type Culture Collection (ATCC, Manassas, USA).ACHN and CAKI-2 cells were respectively cultured in Minimum Essential Medium (MEM) and McCoy's 5A media.Both of media were supplemented with 10% fetal bovine serum (FBS) (Gibco, Shanghai, China).HLECs were cultured in endothelial cell medium with 5% FBS and 1% matched growth factor (ScienCell, CA, USA).A humidified incubator was used for keeping cell lines mentioned above in the condition at 37°C with 5% CO2. ", "section_name": "Cell culture", "section_num": null }, { "section_content": "Briefly, according to the manufacturer's instructions, biotinylated MIR503HG and antisense sequences were pulldown of proteins by using Magnetic RNA-Protein Pull-Down kit (Thermo Fisher Scientific, USA).RNAs were transcribed in vitro transcription with T7 and SP6 RNA Polymerase and the purified transcribed biotinylated RNAs were conjugated with magnetic beads, the conjugation was next incubated with prepared lysates at 4 °C for 24h.Beads were washed briefly to get proteins, protein products were detected by Mass spectrometry and western blotting subsequently. ", "section_name": "RNA pull-down assays", "section_num": null }, { "section_content": "RIP assays were performed to identify the RNA-binding protein by using a MagnaRIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer's protocols.The corresponding pRCC cells lysates were immunoprecipitated with magnetic beads conjugated with anti-H2A.Z antibody or negative control (anti-IgG) at 4 °C overnight.Phenol-chloroform was used to extract the precipitated RNAs.Finally, the eluted RNA was analyzed by qRT-PCR. ", "section_name": "RNA immunoprecipitation (RIP) assays", "section_num": null }, { "section_content": "ChIP assay was performed with EZ-Magna ChIP A/G (17-10086, Millipore, MA, USA) according to the manufacturer's protocol.The enrichment of the H3K27me3, H2A.Z on the promoter region of NOTCH1 was determined by using anti-H3K27me3 and anti-H2A.Z (Abcam, USA).As a negative control, normal mouse IgG were also used for immunoprecipitation.ChIP-derived DNA would be quantified using real-time qPCR analysis. ", "section_name": "Chromatin immunoprecipitation (ChIP) assays", "section_num": null }, { "section_content": "The pRCC cells were previously cultured for 24 h.The conditioned medium was then collected from different groups.HLECs were resuspended in collected conditioned medium and cultured for 12h in 96-well plates (IBIDI, German) placed with 10 μl Matrigel (BD Biosciences, USA).Using inverted fluorescence microscopy to observe tube formation, the formed lymphatic vessels were analyzed and counted by Image J software (NIH, USA). ", "section_name": "Tube formation assay", "section_num": null }, { "section_content": "Cells were cultured on glass cover slips, and IF analysis was then performed as follow: pRCC cells were fixed with 4% formaldehyde for 10 min, and permeabilized with prepared 0.5% Triton X-100 for 15 min at room temperature.Blocked in blocking buffer (TBS with 0.05% tween 20) containing 10% goat serum and 1% BSA for 2 h at 25°C.The slides were then incubated with primary antibody at 4°C overnight, and then subjected to the corresponding secondary antibody incubation for 1h.After incubation, three washes with PBS were performed.Nuclei were stained with 4 ′ ,6-diamidino-2-phenylindole for 10-15 min.Images were taken with confocal microscopy (Leica Microsystems, Wetzlar, Germany).All antibodies used were listed in Table 6. ", "section_name": "Immunofluorescence (IF) staining", "section_num": null }, { "section_content": "Extractions of total RNA from samples were conducted through the TRIZOL Reagent (Life Technologies).Under manufacturers' instructions, RNA was reversely transcribed into cDNA with Color Reverse Transcription Mix (EZBioscience), which was further quantified by qPCR assays to determinate the relative expression level of target gene with Color SYBR Green qPCR Master Mix (EZBioscience).And 2-∆CT method was conducted to evaluate the relative expression among genes.GAPDH were determined as internal control.All the primers used were listed in Table 7. ", "section_name": "RNA extraction and qRT-PCR assay", "section_num": null }, { "section_content": "Genomic DNA was extracted and analyzed from pRCC tissue and paired adjacent normal tissues.The MSP primers were designed from MethPrimer 2.0.purified DNA was exposed to bisulfite with a Bisulfte Conversion Kit (Tiangen, China) according to the manufacturer's protocol.After modified DNA is eluted, a nested, two-stage PCR method were followed to conduct the methylation-specific PCR.Amplified PCR products were identified by agarose gel electrophoresis (1.5%) and visualized with GelRed. ", "section_name": "Methylation-specific PCR (MSP-PCR)", "section_num": null }, { "section_content": "Methylated CpG sites distribution was analyzed in MIR503HG via BGS.Genomic DNA was firstly extracted from frozen pRCC samples and paired adjacent normal tissues.According to the manufacturer's protocol of Epitect Fast Bisulfite Conversion Kits (Qiagen, Germany), genomic DNA was bisulfite modified.methylated cytosine residues were retained as cytosine, wheras the unmethylated cytosine residues were converted to thymine at original sites.BGS assay was then performed to detect the distribution of methylated CpG sites of MIR503HG in each specimen. The CpG islands of MIR503HG promoter region were predicted by MethPrimer 2.0 (http:// www.urogene.org/cgi-bin/methprimer/methprimer.cgi)and EMBOSS database (http://emboss .bioinformatics.nl/cgi-bin/emboss/cpgplot) and 13 CpG sites were selected from MEXPRESS (https://mexpress.be/). ", "section_name": "Bisulfite genomic sequencing (BGS)", "section_num": null }, { "section_content": "4-to-6-week BALB/c nude mice were housed and fed under specific pathogen-free conditions.Luciferase stable expressing cells (1 × 10 6 cells per mouse, mixed with Matrigel, 1:1) were injected orthotopically into capsule of left kidney. We used bioluminecent imaging system (NightOWL II, LB983, Berthold Technologies, Germany) to monitor the primary tumor and potential metastases after abdominal injection of D-Luciferin at different time points (4, 8 and 12 weeks).Animal experiments were in accordance with guidelines of Renji Hospital for the use of animal in cancer research. ", "section_name": "Model of in vivo metastasis", "section_num": null }, { "section_content": "Fresh pRCC samples obtained from two patients underwent surgery at Renji Hospital were implanted into the left flank as subcutaneous tumor in four-week-old anaesthetized immune deficient mice.All mice were observed for maximum 120 days and the tumors from PDXs were analyzed by HE&IHC and qRT-PCR.Animal experiments were in accordance with guidelines of Renji Hospital for the use of animal in cancer research. ", "section_name": "Patient derived xenograft model", "section_num": null }, { "section_content": "The lentiCRISPR vectors containing single-guide RNAs targeting HNRNPC were designed by Beyotime.Following the instruction, sgRNAs were transfected into pRCC cells (CAKI-2 and ACHN) in order to construct HNRNPC knockout (KO) pRCC cells.The efficiency of sgRNA were evaluated by qRT-PCR and western blotting. ", "section_name": "CRISPR-Cas9-mediated gene deletion", "section_num": null }, { "section_content": "Immunohistochemistry (IHC), in situ hybridization (ISH) analysis, subcellular fractionation assays, dual-luciferase reporter assays, enzyme-linked immunosorbent assay (ELISA), lentivirus infection and cell transfection, transwell assays and woundhealing assays were provide in Supplemental Methods. ", "section_name": "Further applied methods", "section_num": null }, { "section_content": "The clinical association of MIR503HG in pRCC was revealed through GEPIA database (http://gepia.cancer-pku.cn/index.html).R language (version 4.0.0,https://www.R-project.org) was used for graphic plotting with following packages: ggplot2, Complex Heatmap, and maftools.Kaplan-Meier analysis was carried out and log-rank analysis evaluate the survival rate of pRCC patients.All date performed at least in triplicate were presented as mean ± standard deviation.One-way ANOVA was used for data comparison when comparing 3 or more experimental groups, with Dunnett's post hoc test for comparing all the groups against the same control group, or Tukey's post hoc test or least significant difference for multiple comparisons with the same n in all groups.Differences among two experimental groups (numeric variables) were evaluated unpaired Student's t-test.Categorical variables were compared by Chi-square (χ2) test or Fisher's exact test.All tests were two-tailed and significant values were set at P < 0.05, P < 0.01, *P < 0.001. ", "section_name": "Bioinformatics and statistical analysis", "section_num": null }, { "section_content": "Summarily, we propose a model that downregulated lncRNA MIR503HG promotes lymphatic metastasis through two pathways: influencing the histone modifications to alter the gene expression of NOTCH1 and upregulating HNRNPC to maturate NOTCH1 mRNA.Additionally, MIR503HG may enable us to suppress pRCC-associated lymphatic metastasis, targeting this newly identified signal pathway is worthwhile in clinic. ", "section_name": "Conclusion", "section_num": null } ]	[ { "section_content": "This work was financially supported by the Natural Science Foundation (grant numbers 81972369, 82173214), Clinical Research Incubation Project of Renji Hospital (RJPY-DZX-004, PYIII20-15), The Young Scholar of Cheung Kong Scholars Program (2022) and the innovative research team of high-level local universities in Shanghai, Hospital-pharma Integration Project on Innovative Achievement Translation (SHDC2022CRD022), Standardized Management Research Project of Shenkang Hospital Development Center (SHDC22021202).Experiments were performed following the Good Clinical Practice and the Declaration of Helsinki under protocols approved by the Ethics Borad of Renji Hospital (KY2022-185-B). ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "All data are included in the manuscript.Raw data would be available upon request. Wei Zhai: orcid.org/0000-0001-6274-6404. ", "section_name": "Data Availability Statement", "section_num": null }, { "section_content": "All data are included in the manuscript.Raw data would be available upon request. ", "section_name": "Data Availability Statement", "section_num": null }, { "section_content": "Wei Zhai: orcid.org/0000-0001-6274-6404. ", "section_name": "ORCID", "section_num": null }, { "section_content": "The authors have declared that no competing interest exists. ", "section_name": "Abbreviations", "section_num": null }, { "section_content": "", "section_name": "Abbreviations", "section_num": null }, { "section_content": "The authors have declared that no competing interest exists. ", "section_name": "Competing Interests", "section_num": null } ]
10.3390/curroncol30020148	Prediction of Clinical Outcomes with Explainable Artificial Intelligence in Patients with Chronic Lymphocytic Leukemia	<jats:p>Background: The International Prognostic Index (IPI) is applied to predict the outcome of chronic lymphocytic leukemia (CLL) with five prognostic factors, including genetic analysis. We investigated whether multiparameter flow cytometry (MPFC) data of CLL samples could predict the outcome by methods of explainable artificial intelligence (XAI). Further, XAI should explain the results based on distinctive cell populations in MPFC dot plots. Methods: We analyzed MPFC data from the peripheral blood of 157 patients with CLL. The ALPODS XAI algorithm was used to identify cell populations that were predictive of inferior outcomes (death, failure of first-line treatment). The diagnostic ability of each XAI population was evaluated with receiver operating characteristic (ROC) curves. Results: ALPODS defined 17 populations with higher ability than the CLL-IPI to classify clinical outcomes (ROC: area under curve (AUC) 0.95 vs. 0.78). The best single classifier was an XAI population consisting of CD4+ T cells (AUC 0.78; 95% CI 0.70–0.86; p < 0.0001). Patients with low CD4+ T cells had an inferior outcome. The addition of the CD4+ T-cell population enhanced the predictive ability of the CLL-IPI (AUC 0.83; 95% CI 0.77–0.90; p < 0.0001). Conclusions: The ALPODS XAI algorithm detected highly predictive cell populations in CLL that may be able to refine conventional prognostic scores such as IPI.</jats:p>	[ { "section_content": "Chronic lymphocytic leukemia (CLL) is the most common leukemic disease in Western countries [1].The WHO Classification for 2022 categorized CLL as a mature B-cell neoplasm [2].CLL is diagnosed by the characteristic immunophenotype in multiparameter flow cytometry (MPFC) B-cell panels [3,4].The prognosis of CLL is heterogeneous; some patients will not require treatment and some patients will progress quickly and some transform into high-grade lymphoma (Richter syndrome).Traditionally, the Rai and Binet classifications of CLL were used for clinical staging and to estimate the prognosis based primarily on leukemia burden [5,6].In addition to the disease burden, genetic factors such as TP53 mutation or 17p deletion, 11q deletion, and a complex karyotype indicate a poor prognosis, while deletion of 13q14 and trisomy 12 harbors a favorable prognosis [7][8][9].Furthermore, mutations in the NOTCH1, SF3B1, and BIRC3 genes are associated with shorter survival [10][11][12][13].Expression of CD38 and ZAP-70 on CLL cells has been associated with unmutated IGHV and higher levels of beta2-microglobulin indicating an adverse prognosis [14][15][16][17][18][19][20]. In 2016, the International Prognostic Index for patients with chronic lymphocytic leukemia (CLL-IPI) was introduced [21].Four prognostic subgroups based on five inde-pendent factors (TP53 status, IGHV mutational status, serum beta2-microglobulin concentration, clinical stage, and age) were defined [21].Five-year overall survival ranges from 93.2% (low-risk CLL-IPI) to 23.3% (very-high-risk CLL-IPI) [21]. However, the five parameters of the CLL-IPI or single markers such as CD38 and ZAP-70 may not reflect the genetic, pathophysiological, and prognostic heterogeneity of the CLL at the individual level compared to structures in large and complex datasets. The MPFC of peripheral blood from patients with CLL generates individual and complex high-dimensional data from malignant CLL cells and non-malignant surrounding white blood cells.MPCF data from CLL patients are acquired routinely for diagnostic purposes. Dimensionality reduction techniques such as self-organizing maps (SOM) have already been applied to enhance the interpretation of MPFC data [22,23].Furthermore, the Citrus (cluster identification, characterization, and regression) algorithm can be applied to find cell-type specific differences between groups in MPFC data [24,25]. Our group described the algorithmic population description approach (ALPODS) which is based on explainable artificial intelligence (XAI) and has been used for classification tasks.The XAI provides sample-based explanations of its decisions by visualizing the immune cell populations which were distinctive for bone marrow compared to peripheral blood [26,27].ALPODS delivered disjunct cell populations which can be visualized in usual flow cytometry two-dimensional dot plots of FCS data files.This enables human flow cytometry operators to classify these cell populations by conventional MPCF gating. MPFC data of diagnostic B-cell panels should include information about the individual CLL prognosis and outcome.Therefore, we used the ALPODS algorithm to identify the crucial cell populations that are overrepresented in CLL patients who experienced death or failure of the first line of systemic therapy [26]. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "", "section_name": "Materials and Methods", "section_num": "2." }, { "section_content": "MPFC data of the peripheral blood from 157 unselected patients with CLL diagnosis were re-analyzed for this study and matched to clinical data.MPFC data were acquired for routine diagnostic analysis at the University Hospital Marburg from 2014 to 2020.The study was approved by the local ethics committee in Marburg.Clinical data included the following: CLL-IPI (i.e., TP53 mutation status, IGHV mutation status, age, Binet, beta2microglobulin), sex, ECOG, Richter transformation, treatment, date of death, treatment failure, and last follow-up.In case of incomplete or unknown CLL-IPI parameters, half of the score points of the missing parameters were given.The patients were separated into a group with an inferior outcome and a group with a superior outcome.Patients who died during follow-up and had a failure of the first-line systemic therapy were categorized as TTF 1 (time to first-line treatment failure), that is, inferior outcome.All other patients were classified as TTF 0 which indicated a superior CLL outcome. We used the ALPODS XAI algorithm [26] to identify cell populations in flow cytometry data that were over or underrepresented in patients with the inferior result (TTF 1) or superior outcomes (TTF 0).The predictive value of the XAI populations was compared to the frequency of CD38-positive CLL cells and CLL-IPI on the receiver operating characteristic (ROC) curves.Multiple logistic regression analysis in repeated 10 bootstrap trials was performed for the combination of more than one independent variable to predict dichotomous groups.Therefore, three randomly selected patients from each group (TTF 0 and TTF1) were left out 10 times to test if the results can be generalized for other patients. ", "section_name": "Patients, Data Acquisition, and Processing", "section_num": "2.1." }, { "section_content": "For diagnosing CLL and other B-cell lymphomas we used a B-cell panel which consisted of two tubes with different fluorescence antibody panels.The first tube (T1) included fluorescence antibodies against B-cell antigens (CD19, CD20, FMC7, CD79b, CD23, light chains of kappa and lambda), T-cell antigens (CD3, CD5, CD2, CD7, CD4, CD8), and the activation marker CD38, which has been described to be prognostic for CLL [14].The sec-ond tube (T2) contained B-cell antigens (CD19, CD20, IgM), markers of hairy-cell leukemia (CD103, CD11c, CD25), follicular lymphoma, and high-grade lymphoma (CD10), and additional markers to ensure the diagnosis of CLL (CD43, CD200).The complete antibody panel, clones, and fluorescence dyes are stated in the Supplementary Information (Table S1). Two 5 mL polystyrene FACS tubes with fluorescence antibodies in a dried-down layer (DuraClone-Technology, Beckman Coulter, Krefeld, Germany) were incubated for 15 min at room temperature in 100 µL prewashed peripheral blood.After antibody staining, red cells were lysed in 2 mL of VersaLyse™ (Beckman Coulter, Krefeld, Germany) for 10 min, washed with 3 mL of buffered phosphate saline (PBS Biochrom, Berlin, Germany), and centrifuged with 300× g for 5 min.The cell pellet was resuspended in 500 µL PBS and measured on a Navios Flow Cytometer (Beckman Coulter, Krefeld, Germany).In total, up to 1 × 10 5 cells were acquired. ", "section_name": "Antigen Panel, Flow Cytometry Staining, and Analysis", "section_num": "2.2." }, { "section_content": "The raw flow cytometry data were compensated, and log transformed.Events with very high side scatter (i.e., mainly granulocytes) were excluded to reduce the amount of data that adds little informative value.Thereafter, data were range standardized between zero and 6 based on the adapted Milligan cooper standardization [28,29].From the total number of recorded cell events of each sample, a 1% random data set was drawn.Using this 1% sample for training ALPODS a 1000-fold cross-validation was performed.The populations that were relevant for the distinction of TTF 1 versus TTF 0 were selected from ALPODS and the most important populations were filtered using Cohen's D effect size measure.The computed ABC analysis [30] selected optimal limits for subset division by exploiting the mathematical properties related to the distribution of the items analyzed.ABC analysis divides the data into three disjoint subsets A, B, and C, with subset A comprising very profitable values, i.e., largest data values (\"the important few\"), subset B comprising values where the yield equals the effort required to obtain it, and the subset C comprising of non-profitable values. ", "section_name": "Data Processing", "section_num": "2.3." }, { "section_content": "The graphs and statistics were compiled with Excel 2016 (Microsoft Corporation, Redmond, WA, USA) and in the R package ggplot2 [31] and DataVisualizations [32], Graph-Pad Prism ® Version 9.4.1 (GraphPad Software, San Diego, CA, USA), R (programming language), www.R-project.org(accessed on 6 October 2022). ", "section_name": "Statistics", "section_num": "2.4." }, { "section_content": "", "section_name": "Results", "section_num": "3." }, { "section_content": "From the 157 CLL patients N = 42 had inferior outcomes (death and/or first-line treatment failure) and N = 115 patients did not reach the defined endpoints (superior outcome, TTF 0).The median age of the total cohort was 68 years (range 26-91 years), and 62 (39.5%) of the patients were female and 95 (60.5%) were male.A total of 83 (52.9%) of the patients were diagnosed with Binet A, 24 (15.3%) with Binet B, and 12 (7.6%)with Binet C. Follow-up was in the median 31.5 months (interquartile range 9-65 months).Additional patient characteristics for the total cohort and separated for TTF 1 or TTF 0 are denoted in Table 1.Abbreviations: N = number; CLL = chronic lymphocytic leukemia; IPI = International Prognostic Index; TTF = time to first-line treatment failure; IQR = interquartile range; R = rituximab. ", "section_name": "Patient Characteristics", "section_num": "3.1." }, { "section_content": "Standardized flow cytometry data and outcome group (TTF 1 or TTF 0) were used as input information for the ALPODS algorithm.ALPODS identified N = distinctive cell populations in the MPFC data which were overrepresented (N = 14/17) or underrepresented (N = 3/17) in the TTF 1 patients' cohort.Seven out of 17 populations were identified in the first tube (T1) of the diagnostic flow cytometry B-cell panel.Ten out of 17 populations were identified in the second tube (T2) of the B-cell panel.The workflow is depicted in Figure 1. Mann-Whitney U test and ROC analysis were performed to detect the most predictive XAI populations for inferior outcomes.The results were listed in Table 2. XAI populations with a significant predictive ability for the outcome (TTF 1 vs. TTF 0) were verified for their prognostic value in patients with high IPI (≥4) compared to patients with low IPI (≤1) (Supplementary Information, Table S2).XAI populations with a significant predictive value for the outcome (TTF 1 vs. TTF 0) and the prognosis (IPI low vs. IPI high) were T1C0011, T1C0016, T2C0004, and T2C0018 (bold script in Table 2).Among these populations, solely T1C0016 had a higher frequency in the patients with a good outcome (TTF 0; mean 13.51% vs. 4.91%; SE of difference 1.83) and good prognosis (IPI ≤ 1; mean 12.50% vs. 5.37%; SE of difference 2.42).Mann-Whitney U test and ROC analysis were performed to detect the most p tive XAI populations for inferior outcomes.The results were listed in Table 2. XAI lations with a significant predictive ability for the outcome (TTF 1 vs. TTF 0) were v In ROC curve analysis, T1C0016 had the highest predictive ability for the outcome of all XAI populations.It should be noted that only the T1C0016 population had the same predictive value as the CLL-IPI score (Table 2; both: AUC 0.78; 95% CI 0.70-0.86).Furthermore, the frequency of CD38-positive CLL cells had a lower predictive ability (AUC 0.66; 95% CI 0.57-0.76,p = 0.0018) than all verified XAI populations TC0011 (AUC 0.76; 95% CI 0.68-0.84,p < 0.0001), T1C0016 (AUC 0.78; 95% CI 0.70-0.86,p < 0.0001), T2C0004 (AUC 0.69; 95% CI 0.58-0.80,p = 0.0002), and T2C0018 (AUC 0.73; 95% CI 0.63-0.82,p < 0.0001). ", "section_name": "Cell Populations Identified by ALPODS", "section_num": "3.2." }, { "section_content": "The 17 XAI populations in combination had a predictive ability of 0.95 AUC (95% CI 0.91-0.98;p < 0.0001) for TTF using multiple logistic regression analysis (Figure 2A), which was significantly higher than IPI (p = 0.0008; Hanley-McNeil test).Restriction on the four populations of XAI (that is, T1C0011, T1C0016, T2C0004, and T2C0018), which were verified to be predictive of IPI, resulted in a lower diagnostic ability of 0.87 AUC (95% CI 0.80-0.93;p < 0.0001) (Figure 2B), but still higher than the conventional IPI (AUC 0.87 vs. 0.78) in this patient cohort, although the difference did not reach statistical significance (p = 0.0771; Hanley-McNeil test). ", "section_name": "EER REVIEW", "section_num": null }, { "section_content": "", "section_name": "EER REVIEW", "section_num": null }, { "section_content": "The ALPODS algorithm calculated FCS data files that can be depicted with co tional two-dimensional flow cytometry dot plots.Therefore, XAI populations can be and analyzed by a human flow cytometry expert.The population T1C0011 has be cated within the CLL cells while T1C0016 consisted of CD4+ T cells (Figure 3A).T2 represented nearly exclusively a subset of CLL cells (Figure 3B) and T2C0018 was ture of a CLL cell subset (higher fraction) and a T and NK cell subset (lower fractio ", "section_name": "Identification of the XAI-Populations", "section_num": "3.3." }, { "section_content": "The ALPODS algorithm calculated FCS data files that can be depicted with conventional two-dimensional flow cytometry dot plots.Therefore, XAI populations can be gated and analyzed by a human flow cytometry expert.The population T1C0011 has been located within the CLL cells while T1C0016 consisted of CD4+ T cells (Figure 3A).T2C0004 represented nearly exclusively a subset of CLL cells (Figure 3B) and T2C0018 was a mixture of a CLL cell subset (higher fraction) and a T and NK cell subset (lower fraction). Interestingly, the most relevant cell population for the outcome (T1C0016) in CLL was not part of the malignant cells but consisted of T helper cells, which were overrepresented in patients with a favorable outcome.This observation led to the question of whether increased CD8+ T cells were predictive of an inferior outcome.Indeed, we found that the XAI population T1C0023 consisted of CD8+ T cells (Supplementary Information, Figure S1).T1C0023 was significantly more abundant in patients with an inferior prognosis (IPI ≥ 4 mean 4.30% vs. IPI ≤ 1 mean 0.48; SE of difference 0.77; p < 0.00461).However, in the ROC analysis, population T1C0023 was not able to classify between TTF 1 and TTF 0 on its own (AUC 0.53; 95% CI 0.41-0.65;p = 0.5573). ", "section_name": "Identification of the XAI-Populations", "section_num": "3.3." }, { "section_content": "The ALPODS algorithm calculated FCS data files that can be depicted with conventional two-dimensional flow cytometry dot plots.Therefore, XAI populations can be gated and analyzed by a human flow cytometry expert.The population T1C0011 has been located within the CLL cells while T1C0016 consisted of CD4+ T cells (Figure 3A).T2C0004 represented nearly exclusively a subset of CLL cells (Figure 3B) and T2C0018 was a mixture of a CLL cell subset (higher fraction) and a T and NK cell subset (lower fraction). ", "section_name": "Identification of the XAI-Populations", "section_num": "3.3." }, { "section_content": "Besides T1C0016 (CD4+ T cells) and T1C0023 (CD8+ T cells), most of the XAI populations were CLL subsets (Tube 1: T1C0011, T1C0012, T1C0017, T1C0019, and T1C0020; Tube 2: T2C0002, T2C0004, T2C0009, T2C0010, T2C0014, T2C0018, and TC0020).The most crucial CLL subsets were T1C0011, T2C0004, and T2C0018, which are shown in Figure 4. Additionally, the median levels of antigen expression and scatter height for relevant subpopulations of CLL cells were compared to the median antigen expression of CLL cells from the average patient in the cohort using a heat map.(Figure 4A,B). It is noteworthy that the populations T1C0011 and T2C0002 showed a decreased forward scatter expression and a decreased antigen brightness compared to mean CLL cells.Visualized on flow cytometry forward scatter, these populations were located partly in the area of dead and apoptotic cells CLL cells (Figure 4C).This finding suggests that a higher frequency of dead and apoptotic CLL cells is associated with a worse prognosis and outcome.In contrast, the CLL subsets T2C0004, T2C0014, T2C0018, and T2C0020 showed higher antigen expression and scatter light profile than mean CLL cells.In summary, CLL cells with small cell volume (low forward scatter) were overrepresented in MPFC data independent of the B-cell panel tube and indicated a poor outcome.On the contrary, subsets of CLL cells with large cell volumes (high forward scatter) also indicated poor outcomes. Besides T1C0016 (CD4+ T cells) and T1C0023 (CD8+ T cells), most of the XAI populations were CLL subsets (Tube 1: T1C0011, T1C0012, T1C0017, T1C0019, and T1C0020; Tube 2: T2C0002, T2C0004, T2C0009, T2C0010, T2C0014, T2C0018, and TC0020).The most crucial CLL subsets were T1C0011, T2C0004, and T2C0018, which are shown in Figure 4. Additionally, the median levels of antigen expression and scatter height for relevant subpopulations of CLL cells were compared to the median antigen expression of CLL cells from the average patient in the cohort using a heat map.(Figure 4A,B). ", "section_name": "Characterization of Predictive Subsets within CLL Cells", "section_num": "3.4." }, { "section_content": "The XAI method ALPODS identified 17 cell populations that were effective at predicting outcome.However, correct manual gating of these populations without using ALPODS is sophisticated, especially for the CLL subsets.Exceptions were the populations T1C0016 and T1C0023, which compromised CD4+ T cells and CD8+ T cells, respectively.Both populations were easy to gate manually in flow cytometry dot plots.Therefore, we tested whether T1C0016 (CD4+ T cells) and T1C0023 (CD8+ T cells) add predictive value to IPI and CD38-positive CLL cells.Multiple logistic regression was performed for this four-factor model (Table 3).Odds ratio (OR) >1 favored inferior outcome and <1 favored superior outcome. In the four-factor model, T1C0023 (OR 1.14; 95% CI 0.95-1.77;p = 0.3305) and CD38positive CLL cells (OR 1.01; 95% CI 1.00-1.02;p = 0.1790) were dispensable.IPI and T1C0016 (two-factor model with IPI) showed only slightly lower predictive value than the four-factor model (0.84 AUC; 95% CI 0.77-0.91;p < 0.0001 vs. 0.83 AUC; 95% CI 0.77-0.90;p < 0.0001).In the two-factor model (IPI and T1C0016) IPI can be replaced by T1C0023 with an acceptable diagnostic ability (0.79 AUC; 95% CI 0.71-0.87;p < 0.0001).Abbreviations: OR = odds ratio; TTF = time to first-line treatment failure; AUC = area under curve; CI = confidence interval, IPI = International Prognostic Index. ", "section_name": "Clinical Significance", "section_num": "3.5." }, { "section_content": "In this single-center study, we analyzed immunophenotypes of 157 CLL patients employing an explainable AI (XAI).The XAI identified 17 cell populations in MPFC data which could in combination predict the clinical outcome of CLL with a higher ability than CLL-IPI or the frequency of CD38-positive CLL cells.Most of the 17 cell populations were located completely or in part within the abundant CLL population.However, some cell populations were non-malignant.For example, the T1C0016 population consisted of CD4+ T cells entirely and was underrepresented in patients with a poorer outcome.T1C0016 (CD4+ T cells) was the best single classifier for the outcome of the 17 XAI-identified cell populations.In contrast, T1C0023 compromised CD8+ T cells that were overrepresented in patients with inferior outcomes. T cells in CLL have been described as dysregulated.CD4+ T cells and CD8+ T cells in patients with CLL deviate from healthy individuals by the accumulation of memory T cells and loss of naïve T cells, increased expression of immune checkpoint receptors (i.e., PD1, TIGIT, CTLA-4), and increased activation [33][34][35][36][37][38].Inversion of the CD4/CD8 ratio is typical for CLL [39][40][41][42].Furthermore, Elston et al. showed that patients with a CD4/CD8 ratio >1 have better overall survival and progression-free survival [34].This is in line with our findings that CD4+ T cells indicated a good outcome and CD8+ T cells an adverse outcome.It would be of interest in further studies to determine which subset of CD4+ cells plays the most significant role in favorable outcomes for patients with CLL.Gating of CD4+ T cells and CD8+ T cells is simple in contrast to XAI populations of CLL subsets and transferrable to other flow cytometry panels that include antibodies against CD4 and CD8.For this reason, we developed a simplified approach to predict the outcome in CLL by the combination of IPI and CD4+ T cells or CD4+ T cells and CD8+ T cells.Both two-factor models discriminate between inferior and superior outcomes in more than 80% of the CLL cases. In addition to diagnostic ability, the XAI populations provided insight into the immunopathology of CLL.For example, we showed that CLL subsets that predict inferior outcomes were small apoptotic/dead CLL events (T1C0011, T2C0002) with low forward scatter.These results are in line with Witkowska et al. and Jahrsdörfer et al. who described that spontaneous in vitro apoptosis of CLL cells correlated with disease progression and cytogenetics with worse prognosis [43,44]. On other hand, CLL subsets with high forward and side scatter (T2C0004, T2C0014, T2C0018, and T2C0020) were associated with adverse outcomes as well.Forward and side scatter correlates with bigger cell size and internal complexity and suggests the prognostic importance of prolymphocytes in CLL.Oscier et al. described that prolymphocytes >10% in CLL are associated with shorter OS and PFS [45]. There are limitations in this study, as some patients did not have complete data for all components of the IPI.In these cases, a half-point score was assigned, which may result in the inaccurate categorization of some patients.A larger patient cohort is warranted to validate our discoveries in multivariate Cox regression models.This would also allow a better clue, if the results are independent of different treatment strategies.Furthermore, an analysis of other prognostic markers for CLL, such as CD49d and ZAP-70, would strengthen the conclusions of the study [46].However, in addition to the established markers that have been shown to have prognostic value in CLL, our XAI study provides new insights into the prognostic factors related to the immunology of CLL and the non-malignant, reactive immune system. ", "section_name": "Discussion", "section_num": "4." }, { "section_content": "The ALPODS XAI algorithm identified and described highly predictive immune cell populations related to outcomes in CLL.In particular, CD4+ T cells were identified as the best single classifier and improved the predictive ability of CLL-IPI.These findings should be further refined with a different immunophenotyping panel and an independent patient cohort. ", "section_name": "Conclusions", "section_num": "5." } ]	[ { "section_content": "We thank Andrea Gruen, Ute Niebergall, and Ute Meissauer for their technical assistance. ", "section_name": "Acknowledgments:", "section_num": null }, { "section_content": "Funding: This work was supported in part by the Deutsche José Carreras Leukämie-Stiftungunder the Grants AH 06-01, 15 R/2020 and in part by a research grant from the University Medical Center Giessen and Marburg (UKGM). ", "section_name": "", "section_num": "" }, { "section_content": "The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/curroncol30020148/s1,Table S1: Fluorochrome, antigens, and antibody clones; Table S2: IPI prediction of the XAI populations; Table S3: Multicollinearity analysis of the four-factor model; Figure S1: T1C0023 (CD8+ T cells).; Figure S2: Kaplan-Meyer curve. ", "section_name": "", "section_num": "" }, { "section_content": "Data Availability Statement: Not applicable. ", "section_name": "", "section_num": "" }, { "section_content": "The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Philipps University (ek_mr_19_10_2020). Informed Consent Statement: Patient consent was waived because flow cytometry data were acquired and recorded for routine diagnostic purposes and data were anonymized and retrospectively analyzed. The authors declare that they have no conflicts of interest.The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. ", "section_name": "Institutional Review Board Statement:", "section_num": null }, { "section_content": "The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Philipps University (ek_mr_19_10_2020). Informed Consent Statement: Patient consent was waived because flow cytometry data were acquired and recorded for routine diagnostic purposes and data were anonymized and retrospectively analyzed. ", "section_name": "Institutional Review Board Statement:", "section_num": null }, { "section_content": "The authors declare that they have no conflicts of interest.The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.18632/oncotarget.15180	Idelalisib and bendamustine combination is synergistic and increases DNA damage response in chronic lymphocytic leukemia cells	Idelalisib is a targeted agent that potently inhibits PI3Kδ which is exclusively expressed in hematological cells. Bendamustine is a well-tolerated cytotoxic alkylating agent which has been extensively used for treatment of chronic lymphocytic leukemia (CLL). Both these agents are FDA-approved for CLL. To increase the potency of idelalisib and bendamustine, we tested their combination in primary CLL lymphocytes. While each compound alone produced a moderate response, combination at several concentrations resulted in synergistic cytotoxicity. Idelalisib enhanced the bendamustine-mediated DNA damage/repair response, indicated by the phosphorylation of ATM, Chk2, and p53. Each drug alone activated γH2AX but combination treatment further increased the expression of this DNA damage marker. Compared with the control, idelalisib treatment decreased global RNA synthesis, resulting in a decline of early-response and short-lived MCL1 transcripts. In concert, there was a decline in total Mcl-1 protein in CLL lymphocytes. Isogenic mouse embryonic fibroblasts lacking MCL1 had higher sensitivity to bendamustine alone or in combination compared to MCL1 proficient cells. Collectively, these data indicate that bendamustine and idelalisib combination therapy should be investigated for treating patients with CLL.	[ { "section_content": "Chronic lymphocytic leukemia (CLL) is a malignancy that is driven by active B-cell receptor (BCR) pathway.The primary BCR down-stream network includes two pivotal enzymes, Bruton's tyrosine kinase and phosphatidyl inositol three kinase (PI3K).Both BTK and PI3K isoform delta are selectively expressed in hematopoietic cells specifically normal and malignant B-cells such as CLL lymphocytes.This exclusive expression provided an impetus to design potent and selective inhibitors of these two proteins.Ibrutinib, a BTK poison and idelalisib a PI3K delta antagonist were tested in CLL and demonstrated on-target effect during preclinical experimentations and efficacy during clinical trials.Both drugs were FDA approved for patients with CLL. As described above, PI3K delta isoform is pivotal in the BCR axis [1] and is selectively blocked by idelalisib [2].Idelalisib promotes apoptosis in CLL by disrupting molecular pathways related to BCR signaling.Furthermore, idelalisib blocks signals from the microenvironment, tumor cell and microenvironment interactions, and mitigates pseudo-emperipolesis [3][4][5][6].Clinically, as a single agent during phase I investigations, the drug has been well-tolerated and has prolonged survival of patients with CLL [2].In combination with rituximab, during phase II clinical trial, the efficacy trend was maintained and this resulted in approval of the drug for relapsed/refractory CLL disease [1,2,7,8]. In the front-line setting, the idelalisib and rituximab combination was tested for older patients with CLL [9].Similarly for another study, treatment-naïve CLL patients were treated with idelalisib for two months as monotherapy followed by combination with ofatumumab Research Paper [10].In both these studies, responses were observed however with a toxicity profile which was much worse than what was observed in the relapsed/refractory disease.The toxicity was identified as immune-mediated and hence was more pronounced in front-line treatment [10,11]. From phase I, phase II in previously treated CLL, and phase II in newly-diagnosed CLL, a few points emerged.First, while the drug was well-tolerated during earlier studies, in treatment naïve patients, there was unacceptable toxicity profile.Second, responses were mostly partial and complete remissions were limited.Third, in both cohort of patients (previously untreated or treated), similar to other BCR pathway antagonists, there was egression of CLL cells from lymph node which remained in peripheral blood.Finally, there is a need to combine idelalisib with CLL-specific agents that may result in deeper responses without much untoward toxicity. Chlorambucil, cyclophosphamide, and bendamustine are three alkylating agents that have been used for decades for treatment of CLL.Of these three, regarding potency in the clinic, the cyclophosphamide is the strongest, followed by bendamustine, and chlorambucil.However, cyclophosphamide results in high untoward toxicity, a feature not favorable to combine with idelalisib.Bendamustine not only possesses alkylating agent properties but is also well-tolerated and FDA approved for patients with CLL along with established treatment recommendations.[12,13]. Previously, we have demonstrated utility of bendamustine in preclinical setting for primary CLL cells and shown synergistic or additive interactions with fludarabine [14].Importantly, both p53 positive and p53 mutated CLL responded similarly to bendamustine [15,16].The mechanism of CLL lymphocyte death elicited by bendamustine was due to DNA damage and repair response.Because PI3K inhibitors, including idelalisib also result in DNA damage, we postulated that combination of idelalisib to bendamustine may result in additive or synergistic cytotoxicity due to enhanced DNA damage.When used alone, bendamustine was more active in suspension cultures of CLL but stromal microenvironment protected CLL cells from bendamustine-mediated cytotoxicity [14].One of the primary pathways of microenvironment-induced CLL cell survival is BCR axis that includes the PI3K/Akt cassette.Because idelalisib mitigates the BCR nexus, we hypothesized that microenvironment-induced resistance to bendamustine cell death of CLL lymphocyte may be abrogated with idelalisib addition. In the current project, we tested that idelalisibmediated suppression of BCR signaling would sensitize CLL cells to bendamustine, and this mechanism-based combination may lead to a synergistic interaction.We evaluated the cytotoxicity induced by idelalisib or bendamustine alone or in combination in primary CLL cells and the impact of this combination on DNA damage response and Bcl-2 family survival protein levels which are regulated by PI3K/Akt pathway.Our data demonstrate that this combination is synergistic, and we suggest a mechanism by which idelalisib increased the cytotoxicity of bendamustine. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "Treatment of primary CLL cells with idelalisib alone (Figure 1A), bendamustine alone (Figure 1B), or the combination for 24 hours (Figure 1C; n = 9) revealed that single agent idelalisib induced moderate but statistically significant cytotoxicity (i.e., apoptosis) in a dose-dependent manner, ranging from 4% to 16% (p = 0.002 to < 0.0001 for each concentration).Whereas single agent bendamustine resulted in 6%-33% (p = 0.002 to < 0.0001 for each concentration) cell death.Combination of idelalisib and bendamustine at clinically relevant concentrations resulted in 13% to 49% of apoptosis (p = < 0.0001 for each concentration).At each drug concentration, compared to single agent apoptotic response, there was an increase in the level of cell death when both drugs were combined together. This dose-response profile led us to investigate whether idelalisib and bendamustine combination could be synergistic combination.To test this, we plotted the apoptotic values obtained from Figure 1C and analyzed by the median-effect method using CalcuSyn software.The calculated combination index (CI) was < 0.8 for all the samples (except for 1 sample treated with low concentrations of both drugs) indicating synergy (Figure 1D). ", "section_name": "Idelalisib and bendamustine combination is synergistic", "section_num": null }, { "section_content": "Bendamustine is an alkylating agent known to induce DNA damage response.γH2AX is a prominent marker of DNA damage response [17] and could be measured using flow-cytometry assay as shown in the plots (Figure 2A).CLL primary cells were either untreated or treated with idelalisib alone, bendamustine alone or combination.Control i.e.DMSO only treated CLL lymphocytes showed a minimal positivity for γH2AX.Compared to the control, single agent idelalisib and single agent bendamustine demonstrated increased γH2AX.Furthermore, when both drugs were incubated simultaneously, the combination treatment significantly enhanced DNA damage response, according to both flow cytometry (Figure 2B) and immunoblot analysis (Figure 2C).When CLL primary cells were stimulated with αIgM, there was a slight decrease in DNA damage response (Figure 2C). We further evaluated the effects of these agents on other proteins involved in DNA damage/repair signaling pathways.Compared to the control, bendamustine alone increased phosphorylation of ATM(Ser1981) and Chk2(Thr68), an effect that was further enhanced by combination treatment.Additionally, bendamustine alone and in combination with idelalisib resulted in stabilization of p53 protein, marked by phosphorylation of p53(Ser15).Overall, these observations indicate that idelalisib enhanced the DNA-damage response elicited by bendamustine. ", "section_name": "DNA damage response was enhanced with combination of idelalisib and bendamustine", "section_num": null }, { "section_content": "Consistent with previous studies of PI3Kα and β isoforms, [18] we found that idelalisib-treated cells had significantly less global RNA synthetic capacity than control cells (47%-71% decrease; n = 4; Figure 3A).We next examined which particular mRNA transcripts are being depleted with idelalisib treatment.In CLL, MCL1 and BCL2 are the members of the BCL2 family antiapoptotic proteins, [19].Our results showed that, in unstimulated cells, idelalisib treatment significantly decreased MCL1 mRNA expression (by 30%); Of note, MCL1 mRNA expression was increased by IgM stimulation, but decreased again with idelalisib treatment (Figure 3B; upper panel).In contrast, BCL2 mRNA expression did not change significantly (Figure 3B; lower panel). ", "section_name": "Idelalisib treatment decreases global RNA synthesis and impacts short-lived mRNAs", "section_num": null }, { "section_content": "In order to examine if the transcription results obtained above are in conjunction with translation, we evaluated the protein levels of Mcl-1 and Bcl-2.CLL primary cells from one patient sample (CLL599) was either unstimulated or stimulated with αIgM and treated with 5 µM of idelalisib for increasing time points (30 min, 2 h and 24 h) and the immunoblot technique was carried out to evaluate Mcl-1 and Bcl-2 proteins.Additional (measure of DNA damage response) after treatment with idelalisib, bendamustine, or the combination.Primary cells were treated with DMSO (D), idelalisib (I, 5 µM) alone, bendamustine (B, 20 µM) alone, or both idelalisib and bendamustine (5 µM + 20 µM, respectively) for 24 hours.The cells were then harvested, fixed with ethanol, incubated with goat serum for 1 hour, and probed with the primary antibody γH2AX.Cells were washed and then co-incubated with PI and DNase-free RNase and analyzed for fluorescence signal on flow cytometry for DNA damage response.Fluorescent cells, with an upward shift were marked as γH2AX-positive cells.B.. Data similar to that in Figure 2A were collected from 5 patients with CLL (CLL514, CLL455, CLL327, CLL137, and CLL103).Horizontal bar represents median value.The significance of the differences between treatment groups was determined by a paired 2-tailed Student t-test.C.. Effect of idelalisib, bendamustine, and combination treatment on DNA damage proteins in CLL cells.Primary cells were either unstimulated or stimulated with αIgM (10 µg/mL) for 30 minutes.The cells were then treated with DMSO, idelalisib alone (5 µM), bendamustine (20 µM) alone, or both idelalisib and bendamustine (5 µM + 20 µM, respectively) for 24 hours.Cells were harvested, lysed and analyzed using immunoblot technique to detect the protein levels of phospho-and total-p53, γH2AX, phospho-and total-Chk2, and phospho-and total-ATM.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control for equal protein loading.idelalisib in primary CLL cells.CLL peripheral blood mononuclear cells isolated from patient samples were treated with dimethyl sulfoxide (DMSO) or with idelalisib (5 µM) for 24 hours.The cells were co-incubated with [5,6-3 H]-uridine for 30 minutes before harvesting, and the amount of radioactivity incorporated into the cells was measured using a scintillation counter.Radioactivity was calculated and normalized to the DMSO control.Experiments using 4 patient samples (CLL057, CLL327, CLL455, and CLL081) were performed in triplicate.The significance of the differences between treatment groups was determined by a paired 2-tailed Student t-test.B. Decreased MCL1 mRNA expression, with no change in BCL2 mRNA expression, in idelalisib-treated CLL cells.Primary cells were either unstimulated or stimulated with αIgM (10 µg/mL) for 30 minutes.Cells then were treated with DMSO or idelalisib (5 µM) for 24 hours and total RNA was extracted and quantified.Isolated RNA was analyzed by real-time RT-PCR with primers and probes for MCL1 and BCL2 mRNA transcripts.MCL1 and BCL2 gene expression levels were measured and normalized to the 18S ribosomal RNA as an endogenous control, and each experiment was normalized to the DMSO control.The results are shown for 8 patient samples (CLL075, CLL483, CLL454, CLL293, CLL068, CLL354, CLL653, and CLL516).Each bar represents mean +/-SEM for the 8 patient samples.The p values were calculated using paired t-test.Abbreviations: NS, not significant.www.impactjournals.com/oncotargetsamples (CLL525 (24 h), CLL103 (24 h) and CLL103 (48 h)) were evaluated for the same proteins.Consistent with our mRNA data, idelalisib treatment decreased Mcl-1 protein but not Bcl-2 protein levels (Figure 4A). ", "section_name": "Idelalisib treatment decreases Mcl-1 but not Bcl-2 protein levels in CLL cells", "section_num": null }, { "section_content": "Single agent bendamustine also depleted MCL1 mRNA levels in primary CLL cells (n = 8) however not to the extent of idelalisib induced depletion tested in the same samples (Figure 5A).When the protein levels were evaluated with bendamustine and bendamustine and idelalisib combination, in both unstimulated and αIgM stimulated cells (24 h and 48 h) there was no marked changes in Mcl-1 protein levels (Figure 5B).On same note, there were no significant changes in BCL2 mRNA (Supplemental Figure 1) or protein levels (Figure 5B).Quantitation of immunoblots for Mcl-1 protein revealed that although single agent bendamustine does not alter the Mcl-1 protein levels, this agent in combination with idelalisib does significantly enhance the depletion of this short-lived protein (Figure 5C). ", "section_name": "Effect of single agent bendamustine and in combination with idelalisib on Mcl-1 expression", "section_num": null }, { "section_content": "Mcl-1, an anti-apoptotic protein of Bcl-2 family is considered important for CLL cell survival.In addition, BCR activation enhances Mcl-1 protein levels and inhibition of BCR signaling with kinase inhibitors deplete Mcl-1.Consistently, our current results demonstrate that idelalisib inhibits Mcl-1 transcript and protein levels, with no differential effect with bendamustine.To directly assess the role of Mcl-1 in bendamustine-induced cytotoxicity, we tested the apoptotic response of isogenic MEFs (Supplemental Figures 2 and3) treated with idelalisib, bendamustine, or combination.Bendamustine-(p = 0.021) or combination-treated (p = 0.017) MEFs lacking MCL1 had significantly higher apoptotic cells compared to wildtype MEFs (Figure 6); indicating that the combination regimen potently downregulates Mcl-1 and thus enhance the apoptosis in CLL cells.This differential effect was not observed with idelalisib (p = 0.478). ", "section_name": "Impact of Mcl-1 loss on bendamustine and combination-induced cytotoxicity in MEFs", "section_num": null }, { "section_content": "Bendamustine alone showed a dose-and timedependent cytotoxicity of quiescent CLL lymphocytes (Figure 1B).The primary mechanism of cell death is damage of DNA, genotoxic stress, and apoptosis [14,20].While idelalisib-induced apoptosis was minor (Figure 1A), the couplet of bendamustine and idelalisib resulted in synergistic combination at many different concentrations (Figure 1D).This is in concert with a prior report where combination of idelalisib at 0.5 µM resulted in sensitization of CLL cells to bendamustine (25 µM) induced cell death [15].Mechanistically, we provide two actions of idelalisib to enhance bendamustine's cytotoxic effect in CLL lymphocytes; first is an impact on DNA damage and repair response and second is a depletion of Mcl-1 protein.Down-regulation of CD69, a biomarker in CLL, by idelalisib has been previously shown to be responsible for sensitization of CLL cells to bendamustine [15]. Bendamustine is an established alkylating agent resulting in double strand breaks.It is 4-{5-[bis(2chloroethyl)amino]-1-methyl-2-bezimidazolyl} butyric acid hydrochloride.The nitrogen mustard group of bendamustine is similar to that in chlorambucil [12,21].However, randomized study comparing chlorambucil to bendamustine suggested increased overall response rate with bendamustine [22].Nitrogen mustard induced damage results in monoadducts, biadducts, and intra-and interstrand cross-links in DNA.As a consequence of this damage response, as expected, bendamustine resulted in induction of gammaH2AX (Figure 2A) along with other hallmark features such as Chk2 phosphorylation (Figure 2B).Paradoxically, and not expected, treatment of CLL cells to idelalisib alone also resulted in initiation of DNA damage response (Figure 2A and 2B and unpublished data).Additionally, in combination with bendamustine, idelalisib resulted in stabilization of p53 protein, marked by phosphorylation of p53(Ser15), Chk2 phosphorylation, and induction of gammaH2AX; all markers of DNA damage.Phosphorylation of p53, Chk2, and H2AX are mediated through active ATM.[14,23,24] It is worthy to mention that none of the patient samples studied had 11q or 17p deletion, the sites for ATM and p53, respectively. The Mcl-1 molecule [25] is an important and bonafide prosurvival protein for CLL.[26] However, as we found in our study, Mcl-1 has been shown to be involved in DNA damage and repair.[27] Other investigators have reported that cytotoxic DNA-damaging agents that cause an early apoptosis response lead to enhanced MCL1 gene expression in a p53-independent manner.[27][28][29][30] Particularly, Mcl-1 is linked to regulating cell-cycle progression and is partially mediated through PCNA, interactions with CDK1, and ATR-dependent activation of Chk1 following DNA damage.[31][32][33] Overall, Mcl-1 is highly overexpressed in many human cancers, is manipulated by malignant cells to escape apoptosis regulation, and has a unique role in the DNA damage response. Mcl-1 is a cytosolic protein and in this location it has been established to inhibit endogenous or druginduced apoptosis.During DNA damage response, it is translocated to nucleus [27,[33][34][35].Nuclear Mcl-1 interacts with many DNA repair/damage proteins such as H2AX, Nbs1, Ku70, and co-localizes with 53BP1 and is involved in homologous recombination pathway [35].2B) and tested for γH2AX.This immunoblot was from the same gel as in Figure 2B and hence same GAPDH is used.C. Immunoblots for three patient samples (from Figure 5B) were quantified by measuring the ratios of Mcl-1 and Bcl-2 to GAPDH for idelalisib-and bendamustine-treated samples, and these values were normalized to the DMSO control.Paired t-test was used to determine p values. In addition to Mcl-1, several other pro and antisurvival members of the Bcl-2 family have been shown to participate in DNA repair.Pro-apoptotic protein Bid binds to RPA protein [36].Bcl-2 has been shown to impact DNA double strand break repair [37] by inhibition of recruitment of Mre11 complex to the site of double strand breaks in the DNA [38].Collectively, these reports establish mechanistic role of Bcl-2 family members in DNA damage repair which is in addition to the pro and anti-survival manifestations of these proteins. Both MCL1 transcript and Mcl-1 protein are shortlived due to the presence of ARE-rich regions and PEST domains in transcript and proteins, respectively.Hence, even a short-term block or inhibition in transcription or protein translation results in a decline in MCL1 transcript and Mcl-1 protein levels.Furthermore, the stability of this protein is impacted by several pathways.Erk, a kinase involved in cancer cell survival, phosphorylates Mcl-1 which prevents proteasomal degradation of Mcl-1 [39,40].Proteasomal degradation of Mcl-1 is dependent on phosphorylation of this protein by GSK3β [41].However, phosphorylation of GSK3β by Akt mitigates activity of GSK3β resulting in prolonged presence of Mcl-1 [42].Both Akt and Erk are induced through BCR pathway [43,44].Corollary to this phenomenon, inhibition of BCR axis by PI3K/Akt inhibitors modulates Akt and Erk activities [2,45] which may impact Mcl-1 protein stability and may induce degradation of this protein. Down regulation of MCL1 transcript and protein levels was observed after treatment of CLL cells during in vitro incubations (Figures 3 and4) of CLL cells with idelalisib.In concert to this observation, clinically, treatment of patients with idelalisib showed lowered expression of Mcl-1 protein in circulating CLL lymphocytes after 2, 4, and 12 weeks of idelalisib intake (Yang, Modi unpublished).Other small molecule targeted inhibitors of PI3K [45], BCR pathway [46], and BTK [47] also result in depletion or decrease in intracellular levels of Mcl-1 protein in CLL lymphocytes.Overall, decrease in Mcl-1 protein expression is a pharmacodynamic phenomenon in CLL cells after treatment with BCR pathway inhibitors. The phase II clinical trial of bendamustine and rituximab in relapsed/refractory CLL disease, demonstrated 60% overall response-rate [48].Although the overall response rate and CR rate are lower than the FCR therapy, this regimen is preferred for low untoward toxicity profile.To increase response rates, fludarabine [49], ibrutinib (HELIOS trial; [50]), and idelalisib [51] have been added to the bendamustine and rituximab couplet regimen.The HELIOS trial was a randomized study where bendamustine and rituximab combination was compared with the triplet of bendamustine and rituximab with ibrutinib.Addition of ibrutinib led to significant benefit for overall response rate and progression-free survival.Importantly, the triple combination did not add any cumulative toxicities [50].Similar to this combination, preliminary and interim report also suggested that idelalisib improves progression-free survival when added to the bendamustine and rituximab couplet [51,52].These trial results clearly suggest clinical benefit of adding BCR pathway inhibitor to bendamustine plus rituximab chemoimmunotherapy. In conclusion, we demonstrate that the addition of idelalisib synergistically benefits bendamustineinduced cytotoxicity in CLL lymphocytes.Furthermore, idelalisib-mediated DNA damage response and decline in MCL1 mRNA and Mcl-1 protein levels may in-part be mechanisms of this synergistic cooperation. ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "", "section_name": "MATERIAL, PATIENTS, AND METHODS", "section_num": null }, { "section_content": "Peripheral blood was obtained from CLL patients (Supplemental Table 1) who had given written informed consent in accordance with the Declaration of Helsinki.The study protocol was approved by the Institutional Review Board of The UT MD Anderson Cancer Center.Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation (Atlanta Biologicals, Norcross, GA).Cells (1×10 7 /mL) were cultured in RPMI-1640 medium with 10% autologous patient serum and were freshly used.For in vitro BCR activation, CLL cells were stimulated with polyclonal goat F(ab')2 fragments of human IgM (MP Biomedicals, Santa Ana, CA). ", "section_name": "Patient sample collection", "section_num": null }, { "section_content": "Mouse embryonic fibroblasts (MEFs), wild-type and MCL1 deficient were generously provided by Dr.Joseph T. Opferman at St. Jude Children's Research Hospital (Memphis, TN) [25] Both cell lines are Simian virus (SV40)-transformed and the cells were maintained in Dulbecco modified Eagle medium with L-glutamine (DMEM; Invitrogen) media supplemented with 10% fetal bovine serum (FBS; Invitrogen), Pen/Strep, L-Glut, and non-essential amino acids (NEAA; GIBCO).Presence or absence of Mcl-1 protein in these cells were confirmed by Dr. Opferman's group as well as by our group using immunoblots.Cell lines were periodically tested for Mycoplasma contamination using a MycoTect kit (Invitrogen).All experiments were conducted in cell passages less than 15 and were maintained at a logarithmic growth concentration between 10 5 cells/mL and 10 6 cells/ mL with 80% confluency as determined by a Coulter channelyzer with less than 10% endogenous cell death confirmed by flow cytometry. ", "section_name": "Mouse embryo fibroblast cell line", "section_num": null }, { "section_content": "Idelalisib (GS-1101 or CAL-101) was provided by Gilead Sciences, Inc., (Foster City, CA).Bendamustine hydrochloride was originally obtained from Cephalon (Frazer, PA; now Teva Pharmaceuticals Industries, Ltd., Petah Tikva, Israel) and was later purchased from Selleckchem (Houston, TX).Both drugs were used in micromolar concentrations that were chosen on the basis of reported plasma concentrations of the free drug in patients.[1,2,5,12,14,20] For bendamustine we used generally 20 µM exogenous drug.Bendamustine peak levels are 28 µM [53] or 20 -24 µM at 90 and 120 mg/m 2 [54,55].For idelalisib, we generally used 20 µM.Because more than 84% of idelalisib binds to human plasma proteins [1], only 16% of the free drug is available to the cells during in vitro culture conditions.Hence, exogenous addition of 5 µM idelalisib to in vitro culture may result in 0.8 µM free idelalisib available for activity.Such free-drug levels in plasma are achieved during idelalisib therapy. ", "section_name": "Drugs", "section_num": null }, { "section_content": "For apoptotic assay, primary CLL cells were untreated or treated with idelalisib, bendamustine, or combination and stained with Annexin V and propidium iodide and counted using flow cytometry, as described previously.[56] Cells in all three quadrants (early apoptosis, late apoptosis, and necrosis) were included to obtain percent total cell death. ", "section_name": "Cytotoxicity assays", "section_num": null }, { "section_content": "The cells obtained before and after incubation with drugs were washed with PBS and fixed in 6 mL ice-cold 70% ethanol and analyzed for H2AX phosphorylation on the flow cytometry.The cells were then fixed with 4% fresh paraformaldehyde/PBS (pH 7.4) at room temperature for 10 min.After couple of washes with BSA/PBS, the cells were blocked with 5% goat serum for an hr.This was followed by incubation with anti-phospho-Histone H2AX (Ser139) mouse monoclonal antibody, clone JBW301, FITC conjugate (16-202A; Upstate, Billerica, MA) for 2 hr.The labeled cells were washed and resuspended in PBS containing the counterstain propidium iodide (15 μg/mL) and RNAase (Roche, South San Francisco, CA) (2.5 μg/ mL) and incubated in dark for 5 min before analysis using FACScalibur (BD Biosciences, San Jose, CA).Data were expressed as fold increase of H2AX phosphorylation.In addition to flow cytometry assay, immunoblot assays were also performed to test for H2AX phosphorylation. ", "section_name": "γH2AX staining", "section_num": null }, { "section_content": "Extracts from cell lysates were quantitated for protein concentration using a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), loaded and transferred to nitrocellulose membranes (GE Osmonics Labstore, Minnetonka, MN, USA).Membranes were blocked for 1 h in licor blocking buffer, incubated with primary antibodies overnight at 4 °C against the following: After washing with PBS-Tween-20, membranes were incubated with infrared-labeled secondary antibodies (LI-COR Inc., Lincoln, NE, USA) for an hour, scanned and visualized using LI-COR Odyssey Infrared Imager.[14] Antibodies for specific proteins and their catalog numbers were used and are listed in the table (Supplemental Table 2). ", "section_name": "Immunoblot analyses", "section_num": null }, { "section_content": "Primary CLL cells were either untreated or treated with idelalisib for the indicated time.For the last 30 min, the cells were incubated with [5,6-3 H]-uridine (1.0 mCi/ mL stock; Moravek Biochemicals, Brea, CA), and the radioactive counts were measured by scintillation counter.[14] Each treatment was done in triplicate and data were presented as percent of control where control is timematched untreated CLL cells. ", "section_name": "RNA synthesis assay", "section_num": null }, { "section_content": "CLL lymphocytes were treated with DMSO or with idelalisib alone, bendamustine alone, or two drugs together for 24 hours.TaqMan real-time reverse transcription polymerase chain reaction assay was used to measure BCL2 and MCL1 transcript levels, which were normalized to 18S ribosomal RNA as an endogenous control.[56] ", "section_name": "Transcript level measurements", "section_num": null }, { "section_content": "Paired 2-tailed Student t-tests were performed using Prism-6 software (GraphPad Software, Inc., La Jolla, CA).For combination treatment, fractional analysis was used to determine whether the combination led to less than, equal to, or more than the additive effect on inducing apoptosis.[57] CalcuSyn software (CompuSyn Inc., Paramus, NJ) was used to determine the combination index. ", "section_name": "Statistical analysis", "section_num": null } ]	[ { "section_content": "Authors are thankful to Jill Delsigne for critically editing the manuscript. ", "section_name": "ACKNOWLEDGMENTS", "section_num": null }, { "section_content": "This work was supported in part by grant P01-CA81534 of the CLL Research Consortium from the National Cancer Institute, the Department of Health and Human Services, a CLL Global Research Foundation Alliance grant, and a Sponsored Research Agreement from Gilead. ", "section_name": "FUNDING", "section_num": null }, { "section_content": "V.G. received research funding from Gilead.Other authors do not have a conflict of interest. P.M. designed the experiments, performed the experiments, analyzed the results, and wrote the manuscript.K.B. directed P.M. in the laboratory and reviewed the manuscript.Q.Y. assisted in experimental planning and reviewed the manuscript.M.J.K. and W.G.W. identified patients to obtain peripheral blood samples, provided clinical and patient-related input, and reviewed the manuscript.V.G. conceptualized and supervised the research, obtained funding, analyzed the data, and wrote and reviewed the manuscript. This paper has been accepted based in part on peerreview conducted by another journal and the authors' response and revisions as well as expedited peer-review in Oncotarget. ", "section_name": "CONFLICTS OF INTEREST", "section_num": null }, { "section_content": "V.G. received research funding from Gilead.Other authors do not have a conflict of interest. ", "section_name": "CONFLICTS OF INTEREST", "section_num": null }, { "section_content": "P.M. designed the experiments, performed the experiments, analyzed the results, and wrote the manuscript.K.B. directed P.M. in the laboratory and reviewed the manuscript.Q.Y. assisted in experimental planning and reviewed the manuscript.M.J.K. and W.G.W. identified patients to obtain peripheral blood samples, provided clinical and patient-related input, and reviewed the manuscript.V.G. conceptualized and supervised the research, obtained funding, analyzed the data, and wrote and reviewed the manuscript. ", "section_name": "Authorship contribution", "section_num": null }, { "section_content": "This paper has been accepted based in part on peerreview conducted by another journal and the authors' response and revisions as well as expedited peer-review in Oncotarget. ", "section_name": "Editorial note", "section_num": null } ]
10.1038/srep46135	Notch and Hippo signaling converge on Strawberry Notch 1 (Sbno1) to synergistically activate Cdx2 during specification of the trophectoderm	<jats:title>Abstract</jats:title><jats:p>The first binary cell fate decision occurs at the morula stage and gives rise to two distinct types of cells that constitute the trophectoderm (TE) and inner cell mass (ICM). The cell fate determinant, Cdx2, is induced in TE cells and plays an essential role in their differentiation and maintenance. Notch and Hippo signaling cascades are assumed to converge onto regulatory elements of <jats:italic>Cdx2,</jats:italic> however, the underlying molecular mechanisms are largely unknown. Here, we show involvement of Strawberry Notch1 (Sbno1), a novel chromatin factor of the helicase superfamily 2, during preimplantation development. <jats:italic>Sbno1</jats:italic> knockout embryos die at the preimplantation stage without forming a blastocoel, and Cdx2 is not turned on even though both Yap and Tead4 reside normally in nuclei. Accordingly, Sbno1 acts on the trophectoderm-enhancer (TEE) of <jats:italic>Cdx2</jats:italic>, ensuring its robust and synergistic activation by the Yap/Tead4 and NICD/Rbpj complexes. Interestingly, this synergism is enhanced when cells are mechanically stretched, which might reflect that TE cells are continuously stretched by the expanding ICM and blastocoel cavity. In addition, the histone chaperone, FACT (FAcilitates Chromatin Transcription) physically interacts with Sbno1. Our data provide new evidence on TE specification, highlighting unexpected but essential functions of the highly conserved chromatin factor, Sbno1.</jats:p>	[ { "section_content": "Transcription factors Tfap2c and Gata3 also directly regulate Cdx2, either upstream or downstream of Hippo signaling 5,6 , and cooperation between Yap/Tead4 and Notch1/Rbpj transcriptional activities on Cdx2 in the TE was also reported 7 ; hence the specification of the TE is controlled by a joint action of different signaling cascades and transcription factors. In addition to the cell-type specific actions of transcription factors, ATP-dependent helicase-related factors involved in chromatin remodeling have recently been shown to be essential during embryonic development 8 .For example, the helicases or helicase-related enzymes unwind and/or twist DNA/RNA to alter chromatin structures, which is a prerequisite for subsequent events, such as gene transcription or DNA replication and repair.These helicase-like proteins can be classified into six groups, namely helicase superfamily 1 to 6 (SF1 to SF6), based on their sequences and conserved motifs [9][10][11] .Among them, DExx box helicases and Swi/Snf chromatin remodelers are classified as the SF2 superfamily. Strawberry Notch (Sbno in vertebrates, Sno in Drosophila) is a helicase-related nuclear factor.The N-and C-terminal regions of Sbno/Sno are highly conserved in both vertebrates and invertebrates 12,13 , and these regions contain two characteristic motifs, the DExH box and helicase-c domain, respectively.Based upon these structural features, Sbno/Sno is classified as a helicase-like protein [14][15][16] that belongs to the SF2 superfamily.Nonetheless, the molecular functions of Sbno/Sno, especially from a viewpoint of transcriptional control, remain obscure. Genetic and molecular analyses in fly, worm and fish have revealed that Sbno/Sno is relevant to developmental processes that involve Notch.In Drosophila, sno mutants are embryonic lethal with severely impaired cuticular and nervous system development.In contrast, heat-inducible sno mutants in eclosed flies phenocopy the notch or Su(H) mutant, which shows disrupted ommatidia, fused segments of legs and notched wing margin.More importantly, these phenotypes can be rescued by additional notch or Su(H) 17 .In the developing wing margin, Notch-induced sno regulates expression of wingless, vestigial, cut and E(spl)-m8 12,18 .These lines of evidence suggest that sno acts in the Notch cascade, thereby affecting other signaling pathways, such as Wnt and Hippo 18 , and highlighting its crucial action at the intersection of different signaling pathways.During photoreceptor specification in Drosophila, Sno binds to Su(H) and an F-box/WD40 protein Ebi, which recruit the transcriptional co-repressor SMRTER to keep its direct target Delta inactive.This transcriptional repression is relieved by epidermal growth factor receptor (EGFR) signaling, and this de-repression is proteasome-dependent and accompanied by cytoplasmic translocation of SMRTER.This EGFR pathway-regulated Delta transcription allows transmission of Delta signal to neighboring Notch-expressing cells, a molecular basis for the binary specification of photoreceptor and non-neuronal cone cells 13 .On the other hand, in C. elegans, let765/sno functions upstream of the lin-3/ egf-Ras pathway to regulate vulval development 15 .In zebrafish, Sbno1 also interacts with Su(H), and is involved in neural development 19,20 .These studies indicate that Sbno/Sno acts on different signaling pathways and also in distinct tissue-specific contexts, yet its precise molecular actions are largely unknown. In this study, we analyzed Sbno1 function during mouse development.When Sbno1 is disrupted in mouse, embryonic development is arrested at the preimplantation stage with a loss of expression of TE-specific genes.We found that Sbno1 is required for transcriptional activities of Yap/Tead4 and Notch/Rbpj.Furthermore, Sbno1 is indispensable for transcriptional activation of the Cdx2 TE enhancer, which is regulated by a synergistic action of Yap/Tead4 and Notch/Rbpj.Physical interaction between Sbno1, Yap/Tead4, Rbpj and the FACT complex indicates that Sbno1 regulates activity of these transcription factors on target genes.Our results highlight a critical role of this helicase-related factor on specific gene activation during preimplantation development. ", "section_name": "", "section_num": "" }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Sbno1 in mouse preimplantation embryos.Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analyses revealed that Sbno1 transcripts are present in both oocytes and preimplantation embryos (Fig. 1a).The expression level decreased soon after fertilization, then recovered gradually with cell division (Fig. 1a).In contrast, Sbno1 protein was not detected in the oocyte (Fig. 1b).The first nuclear localization of Sbno1 was detected at low levels in the zygote (Fig. 1b).Robust levels of Sbno1 were observed in the nuclei of preimplantation embryos from the two-cell stage, and this nuclear localization was maintained during cell division and compaction (Fig. 1b).At embryonic day 3.5 (E3.5) the embryo has developed into a blastocyst, which consists of the ICM, outer TE and blastocoel.Sbno1 was detected in the nuclei of both ICM and TE cells (Fig. 1b).Throughout the developmental processes, Sbno1 was observed exclusively in the nucleus, suggesting a nuclear function.Expression patterns of Sbno1 at later stages are shown in Supplementary Fig. 1. We then generated Sbno1 knockout mice by targeting exon 7 of Sbno1, which harbors the N-terminal DExH box region (Supplementary Figs 2 and3).After Cre-mediated recombination, the targeted allele contains a frame-shift, resulting in a premature stop codon.We could not obtain Sbno1 homozygous embryos (Sbno1 Δ/Δ ) from Sbno1 Δ/+ intercrosses at post-implantation stages, indicating embryonic lethality during very early stages of development.To examine further, we collected preimplantation embryos.At E3.5, control heterozygous embryos developed to the blastocyst stage.In contrast, Sbno1 knockout embryos did not form the blastocoel but retained an un-compacted morphology (Fig. 1c,d).When Sbno1 protein was checked by immunohistochemistry, it was absent from the two-cell stage in Sbno1 knockout embryos (Fig. 1f), whereas the signal was clearly evident in Sbno1 Δ/+ embryos from E1.5 to E3.5 (Fig. 1e), confirming the absence of Sbno1 in the knockouts. Next, we performed in vitro embryo culture to observe serial development of Sbno1 knockout embryos from the two-cell to blastocyst stage (Fig. 1g,h, Supplementary movie S1).Sbno1 knockout embryos compacted normally at the morula stage but failed to form the blastocoel, resulting in fragmentation of the embryo 72 hours after initiation of in vitro culture.Extensive cell death then occurred, as shown by TUNEL staining (Fig. 1j).Under these culture conditions, control Sbno1 Δ/+ embryos gave rise to blastocysts, via compaction and formation of the blastocoel (Fig. 1g), and did not show any TUNEL staining (Fig. 1i).In addition, cell proliferation was significantly repressed in the Sbno1 knockout embryos at E3.5, as shown by phospho-Histone H3 staining (Fig. 1k,l).Quantitatively, the phospho-Histone H3 staining was reduced to approximately 20% of that in the control embryos (Fig. 1m).These results indicate that Sbno1 regulates a critical step of the morula-to-blastocyst transition, and that a loss of Sbno1 function results in cell cycle arrest and consequent apoptotic cell death after the 16-cell stage.Consistent with this cell death in the Sbno1 knockouts, Sbno1 knockout embryonic stem (ES) cells could not increase cell number, and intensive cell death occurred (Supplementary Fig. 4), indicating that Sbno1 is essential for cellular survival of ES cells. ", "section_name": "Sbno1 functions during mouse preimplantation development. We first examined expression of", "section_num": null }, { "section_content": "Sbno1 might regulate the expression of genes that are essential for the morula-to-blastocyst transition, we examined expression of genes that mark the differentiation of the ICM and TE in E2.5 control (wild-type), E3.5 control (Sbno1 +/+ or Sbno1 Δ/+ ) and Sbno1 knockout embryos.One of the ICM markers, Oct3/4, was observed equally in both the control and Sbno1 knockout embryos at E2.5-E3.5 (16-cell stage), as revealed by immunohistochemistry (Fig. 2a-c).Again, the Sbno1 knockouts did not develop to the blastocyst stage, yet Oct3/4 levels were maintained, even at E3.5 (Fig. 2c).At E3.5, Nanog levels remained low in the Sbno1 knockouts (Fig. 2f), at the same level as the control at E2.5 (Fig. 2d), whereas they were increased in the ICM of the control at E3.5 (Fig. 2e), indicating that reduced levels of Nanog in the Sbno1 knockouts were due to developmental delay.Consistent with the immunohistochemistry, the semiquantitative RT-PCR analysis with E3.5 embryos revealed that three ICM markers, Oct3/4, Sox2 and Gata6, were expressed at the same level in both controls and knockouts, yet expression of Nanog was decreased solely in the Sbno1 knockouts (Fig. 2m).These data indicate that formation of the ICM was unaffected in the absence of Sbno1, except for the decline of Nanog at E3.5.c,f,i,l).In 16-cell Sbno1 Δ/Δ embryos at E3.5, Oct3/4 and Nanog were expressed at similar levels to those in the 16-cell stage control embryos (a-f).On the other hand, expression of Cdx2, Eomes and Krt8 was not evident in Sbno1 Δ/Δ embryos (a-c,g-l).(m) RT-PCR analysis of E3.5 control and Sbno1 Δ/Δ embryos.Although Oct3/4, Sox2 and Gata6 were expressed at normal levels, expression of Nanog, Cdx2, Eomes and Krt8 was greatly decreased in Sbno1 Δ/Δ embryos.Hprt was used as an internal control.(n-v) Immunohistochemistry of Yap, Cdx2, Tead4 and Rbpj in E2.5/E3.5 control (n,o,q,r,t,u) and E3.5 Sbno1 Δ/Δ embryos (p,s,v).Nuclear localization of Yap in the Cdx2-expressing TE cells was observed in the control embryos (n,o), In Sbno1 Δ/Δ embryos, expression of Cdx2 was very faint but Yap is clearly localized in nuclei (p).Expression of Tead4 and Rbpj in Sbno1 Δ/Δ embryos was as the same as that in the control (q-s,t-v).Semiquantitative RT-PCR analysis showed expression of Yap, Tead4, Notch1 and Rbpj in control and Sbno1 Δ/Δ embryos at E3.5 (w).Uncropped image of gels are shown in Supplementary Fig. 10. In contrast to the ICM markers, Cdx2, an early TE marker, was barely detectable in the Sbno1 knockout embryos at E2.5 and E3.5 (Fig. 2c, Supplementary Fig. 5), whereas Cdx2 was evident in both E2.5 embryos and the TE cells in the control blastocysts at E3.5 (Fig. 2a,b, Supplementary Fig. 5).Although the development of Sbno1 knockout embryos was slightly delayed at E2.5, Cdx2 expression was not turned on even in the 16-cell Sbno1 knockout embryos at E3.5, suggesting that induction of Cdx2 expression is not normal in the absence of Sbno1.Other TE markers, such as Eomesodermin (Eomes) and Keratin 8 (Krt8), whose expression is regulated by Cdx2 21,22 , were also absent in the Sbno1 knockout embryos at E3.5 (Fig. 2g-m).The absence of or reduced expression of Cdx2, Eomes and Krt8 was further confirmed by semiquantitative RT-PCR analysis at E3.5 (Fig. 2m).These results indicate that differentiation of the TE is severely impaired in the absence of Sbno1. Yap and Tead4 interact in the nuclei of the outer cells of the preimplantation embryo 3 , acting upstream in TE differentiation.In cooperation with Yap/Tead4, Notch/Rbpj signaling directs TE fate by regulating Cdx2 transcription 7 .Hence, we checked protein levels of Yap, Tead4 and Rbpj, and found that these essential components were expressed normally with correct nuclear localization (Fig. 2n-w).This strongly suggests that Yap/Tead4 and Notch/Rbpj could not activate Cdx2 in the absence of Sbno1, even though they were correctly localized in the nucleus.This suggests a regulatory role of Sbno1 on the Yap/Tead4 and Notch/Rbpj-mediated transcription of Cdx2.Sbno1 is a nuclear protein; therefore, we characterized its molecular functions as a novel transcriptional regulator. Sbno1 stimulates Yap/Tead and Notch/Rbpj transcriptional activities.Careful inspection of the protein structure of Sbno1 (human, mouse, fly and nematode) and comparison with other factors revealed several key features of Sbno1 as a member of the SF2 family, which includes DExD/H box helicases and Swi2/Snf2 remodelers (Fig. 3a, Supplementary Fig. 3) [23][24][25][26] .Sbno1 proteins are highly conserved among species (human vs mouse 97.6%, vs fly 55.3%, vs nematode 47.4%), and two characteristic features of the SF2 family, namely the DExH box and helicase-c domains, are found in the N-and C-terminal regions of Sbno1, respectively (Supplementary Fig. 3).These distinctive features strongly suggest that Sbno1 might be involved in transcriptional control of gene expression by acting as a chromatin remodeler. In addition to the structural aspect, genetic analyses of strawberry notch (sno), a Drosophila ortholog of Sbno1, suggest that sno positively regulates transcription of its targets to activate Notch (Delta), Wnt (wingless) and Hippo (scalloped, vestigial) signaling pathways 12,13,18,27,28 .These lines of evidence indicate that Sbno1/sno might be a crucial transcriptional integrator acting at the intersection of different signaling cascades. To investigate this possibility, we first made an artificial construct, in which human SBNO1 was fused with a gene encoding the Gal4-DNA binding domain.This binds to Gal4-binding sequence multimerized and inserted upstream of the chicken δ-crystalline minimal promoter and the luciferase reporter 29 .When the Gal4-luciferase assay was performed, Gal4-SBNO1 induced robust activation of transcription (Gal4-SBNO1 Wt, 57.5-fold; Fig. 3b), whereas the Gal4-DNA binding domain alone did not show any effect on luciferase activity (Gal4, 0.8-fold; Fig. 3b), indicating that Sbno1 acts as a potent transcriptional activator.Next, we constructed deletion mutants, by dividing SBNO1 into three regions (N-terminal, Middle and C-terminal parts; Fig. 3a).Gal4 fused with the SBNO1-N terminal region (Gal4-N) retained the luciferase activity, whereas the activator function was lost when the M and C regions were used (Fig. 3b).The N-terminal region harbors the DExH box containing ATPase activity; therefore, we mutated E437, an essential glutamic acid residue for DExH domain ATPase, activity 30,31 to glutamine (Q) (Fig. 3a).As expected from the essential role of ATPase activity in the SF2 family 30,31 , the Gal4-SBNO1-E437Q mutant completely lost luciferase activity (Fig. 3b). To address whether the Sbno1 activity is required for the preimplantation development, we microinjected EGFP-hSBNO1-Wt or -E437Q mutant mRNA to mouse zygotes, and cultured them for 3 days.Contrary to hSB-NO1-Wt, development of most of hSBNO1-E437Q-injected embryos arrested at 8-12 cell stages (Fig. 3c-f), earlier than the Sbno1 knockouts (Figs 1 and2), and this arrest was accompanied by reduction of Cdx2 expression (Fig. 3g).This result indicates that E437 in the DExH domain is crucial for the function of Sbno1 during preimplantation development. Expression of Cdx2 was dramatically reduced in the Sbno1 null embryos; therefore, we speculate that Sbno1 might be involved in the transcriptional control by the Yap/Tead4 and Notch/Rbpj complexes, which act through the Hippo and Notch cascades, respectively, to directly regulate Cdx2 7 .Importantly, Drosophila sno functions on these two signaling networks 18 .To explore this possibility, we analyzed transcriptional control by the Yap/Tead complex, using a reporter that contains eight repeats of the Tead binding site (5′ -GCTGTGGAATGTGTGTC-3′ ) upstream of a minimal δ-crystallin promoter (8xGT-IIc-Luciferase) 32 .When 293 T cells were transfected with this reporter, along with a Yap expression plasmid, robust activation of the luciferase reporter was observed (60.7-fold;Fig. 4a).This activation was repressed by endogenous SBNO1 knockdown (26.0-fold;Fig. 4a), but the reporter without Tead binding sites was unaffected (data not shown).Reduction of SBNO1 protein levels by siRNA was confirmed by western blotting using an anti-SBNO1 antibody (Supplementary Fig. 6).These results indicate a contribution of Sbno1 to Yap/Tead-mediated transcriptional activation. To explore further the function of Sbno1 in Yap/Tead-mediated Hippo signaling, we determined the effects of Gal4-SBNO1 or Gal4-Tead4-mediated transcriptional activation on Yap (Fig. 4b).As expected, in the presence of Yap, the Gal4-Tead4 fusion protein activated the Gal4 reporter robustly, by approximately 8,100-fold, confirming the reliability of this assay.Gal4-SBNO1 alone activated the reporter (68.6-fold), but when Yap was co-expressed, enhancement of activation was observed (approximately 1,400-fold; Fig. 4b). Yap nuclear localization is promoted by escaping cell contact inhibition 32,33 ; therefore, we performed the same experiment with different cell densities (Fig. 4c).Gal4-SBNO1 alone exhibited similar effects, regardless of cell density (113-, 142-and 64-fold activation).In clear contrast to this, when Yap was co-expressed, super-activation of Gal4-SBNO1 (3088-fold) was observed at low cell density, yet this robust activation was largely repressed at high cell density to 343-fold.This strongly suggests that Sbno1 can act in concert with the Hippo pathway, which is known as a sensor of the physical milieu, as represented by cell density 32,34,35 . Involvement of Sbno1 on Notch/Rbpj transcriptional activity was also examined with a TP1-luciferase reporter, which contains 12 copies of the Rbpj binding site, and is activated by Notch intracellular domain (NICD) and Rbpj 36 .When luciferase activities derived from this reporter were measured in 293 T cells, the reporter was activated by an active form of Notch1 (Notch1Δ E, extracellular domain-deleted) 37 , and this transcriptional activation was repressed by co-introduction of SBNO1 siRNA (Fig. 4d), indicating the requirement of Sbno1 for efficient transcription of Notch/Rbpj target genes. The hSBNO1-E437Q mutant lacks function as a transcriptional activator (Fig. 3b); therefore, we speculated that this mutant SBNO1 would act as a dominant negative mutant.To confirm this possibility, we expressed hSBNO1-E437Q along with the Tead or Notch reporter and their effectors, Yap and Notch1Δ E, respectively.Consistent with the SBNO1 siRNA experiments, hSBNO1-E437Q repressed both the Tead and Notch reporters in a dose-dependent manner (Fig. 4e,f).These results revealed that intact Sbno1 DExH box activity is indispensable for the transcriptional activation of Tead and Rbpj by their co-activators, Yap and Notch, respectively. Sbno1 is necessary for normal Cdx2 trophectoderm enhancer activity.In Sbno1 knockout embryos, Cdx2 expression was significantly decreased (Fig. 2).Recently, expression of Cdx2 in the TE has been shown to be regulated by Yap/Tead4 and Notch/Rbpj though binding to sites in the trophectoderm-enhancer (TEE) in the Cdx2 gene 7 .To analyze the roles of Sbno1, we made a new luciferase reporter, in which a short element (47 bp) of the Cdx2 TEE containing both the Tead and Rbpj biding sites was tetramerized and inserted in front of the minimal δ-crystallin promoter (4xCdx2-TEE47bp; Fig. 5a).As expected, Yap and Tead4 synergistically activated 4xCdx2-TEE47bp in 293 T cells (367-fold; Fig. 5b), whereas sole expression of Yap or Tead4 resulted in only mild activation or repression (34-and 0.3-fold, respectively; Fig. 5b).Notch1Δ E alone produced weak activation (3.9-fold, respectively; Fig. 5b).Nonetheless, when Yap and Notch1Δ E or all three effectors were simultaneously introduced, activation of 4xCdx2-TEE47bp increased by 1,287 and 1,404-fold, respectively (Fig. 5b).The activation of Cdx2-TEE was significantly suppressed by knocking-down SBNO1 (Fig. 5b).When the hSB-NO1-E437Q mutant was used instead of knock-down, repression of synergistic activation by Yap, Tead4 and Notch1Δ E was more evident, resulting in approximately 20% activation (Fig. 5c). Similarly to 293 T cells, E14Tg2a ES cells showed synergistic activation of the 4xCdx2-TEE47bp by Yap and Notch1Δ E. This synergism was again significantly suppressed by the hSBNO1-E437Q mutant, whereas the hSBNO1-Wt enhanced the cooperative effect of Yap and Notch1Δ E on the 4xCdx2-TEE47bp in E14Tg2a ES cells (Fig. 5e), although it did not influence to the activities of 4xCdx2-TEE47bp reporter in 293 T cells (Supplementary Fig. 7).These results clearly demonstrate that Sbno1 is an essential component at the convergence of two different signaling cascades, namely Hippo and Notch. As shown in Fig. 4c, transcriptional activation by Yap is dependent on cell density.Recently, the Hippo cascade has been shown to be sensitive to cytoskeletal tension, highlighting Yap as a mechanotransducer 34,35,[38][39][40][41][42] .Likewise, physical force can activate the Notch signaling 43 .Hence, the Notch and Hippo cascades are both sensitive to the physical state of cells.Next, we confirmed whether activation by these two pathways is also sensitive to physical parameters (Fig. 5e) by stretching transfected cells on a silicone membrane.Even at high or low cell density, co-transfection of Yap and Notch1Δ E activated 4xCdx2-TEE47bp robustly, and at the same intensity (713 vs. 651-fold induction).More importantly, the transactivation was super-enhanced when cells were stretched at high cell density (a 713-1764-fold induction), whereas the mechanical stretch at a low cell density had a repressive effect (651-490-fold).These lines of evidence indicate that transcriptional activation of Cdx2 through the TEE is dependent on the physical state of cells. Sbno1 physically interacts with Yap/Tead4 and NICD1/Rbpj.Cooperative regulation of Cdx2 by Sbno1, Yap/Tead4 and Notch/Rbpj suggests that these factors physically interact with each other.To explore this, we carried out co-immunoprecipitation (CoIP) analysis, and found that SBNO1 indeed interacts physically with Tead4, as revealed by co-precipitation of SBNO1 with Tead4 as well as Yap (IP:α -Myc; Fig. 6a).In contrast, when Yap was precipitated by an anti-HA antibody (IP:α -HA; Fig. 6a), only Tead4 was co-precipitated; therefore, interaction between SBNO1 and Yap might only be detected in limited conditions.When Tead4 was precipitated, co-purification of SBNO1 was observed weakly only in the absence of Yap (IP:α -FLAG; Fig. 6a).These data suggest that the majority of Tead4 and Yap forms a complex, and that only a fraction of this complex co-exists with SBNO1.In addition, these data also suggest that interaction between Tead4 and SBNO1 becomes weak in the presence of Yap, despite formation of the Tead4 and Yap complex. We confirmed the interaction between SBNO1 and Rbpj, which was previously reported in Drosophila and zebrafish 13,19 .When SBNO1 was precipitated, both Rbpj and NICD were co-purified, albeit NICD1 co-precipitation was very weak (Fig. 6b).When NICD1 was precipitated, only Rbpj was co-purified (Fig. 6b).Likewise, SBNO1 and NICD were co-precipitated along with Rbpj (Fig. 6b).More importantly, however, interaction of SBNO1 with Rbpj was again attenuated by NICD1, as represented by fainter bands of SBNO1 and Rbpj (Fig. 6b), a similar observation to that of Yap/Tead4 (Fig. 6a).These observations suggest that interaction of SBNO1 with DNA-binding proteins, such as Tead4 and Rbpj, weaken when their co-activators (Yap and NICD1, respectively) arrive in the nucleus and bind to their partners. We next confirmed the physical interaction of Sbno1 and Tead4 in E3.5 embryos by in situ proximity ligation assay (PLA).Consistent with the in vitro CoIP analyses, the PLA signal between Sbno1 and Tead4 proteins was observed in the outer cells where Cdx2 is expressed.As expected, the Yap and Tead4 interaction gave the signal at the same level (Fig. 6c-e), indicating that Sbno1 and Yap/Tead4 make a complex in the TE cells. ", "section_name": "Trophectoderm markers are not induced in Sbno1 knockout embryos. To investigate whether", "section_num": null }, { "section_content": "Yap alone or NotchΔ E alone activated the 4xCdx2-TEE47bp reporter (39-and 3.9-fold, respectively), yet when both Yap and NotchΔ E were co-expressed, this reporter was synergistically and robustly activated (1287-fold).As expected, this activation was repressed by SBNO1 siRNA (664-fold).Synergism between Yap and Tead4 was observed (367-fold); however, expression of Tead4 did not affect the synergistic activation by Yap and NotchΔ E (1287-versus 1404-fold activation).(c) Lack of transcriptional activation of the 4xCdx2-TEE47bp reporter was evident when the SBNO1-E437Q mutant was expressed.(d) Transcriptional activity of the 4xCdx2-TEE47bp reporter was synergistically upregulated by Yap and NotchΔ E in E14Tg2a ES cells, and the SBNO1-E437Q mutant significantly decreased the activity (6.9-and 3.2 fold, respectively).In contrast, SBNO1-Wt increased the Yap and NotchΔ E-induced transcriptional activity (16.8 fold).(e) Synergistic activation of the 4xCdx2-TEE47bp reporter by Yap and NotchΔ E was observed at both high and low cell densities in the absence of mechanical stretch (713-and 651-fold, respectively).In contrast, when cells were stretched, this synergistic activation was super-enhanced to 1764-fold only in the high cell density culture.At low cell density, mechanical enhancement of transactivation was not observed, and was slightly repressed (490-fold).Pictures of cell cultures are shown.Note that cells make mutual contacts at high density, while at low density cells are isolated or clustered in small separated islands of cells.All data are presented as means ± SD. **p < 0.01 versus relevant control. ", "section_name": "(b) Expression of", "section_num": null }, { "section_content": "To gain more insight into the role of Sbno1 in the transcriptional control of Cdx2, we mined a previous high-throughput interactome analysis in HeLa cells 44 , and found SSRP1 and Cxorf26 as interacting partners for SBNO1.Although the function of Cxorf26 is unknown, Ssrp1, a high mobility group (HMG) domain-containing protein, is known to heterodimerize with Spt16 to form the FACT complex.This complex acts as a histone H2A/H2B chaperon to assist progression of RNA polymerase II on its DNA template during transcriptional elongation 45,46 , thereby positively controlling gene expression. To determine whether SBNO1 could be a crucial component of this chaperone machinery, we examined the interaction between SBNO1 and Ssrp1 using the CoIP assay.When SBNO1 or Ssrp1 was precipitated, Ssrp1 and SBNO1 were co-purified, respectively (Fig. 7a).We then further probed the relationship between SBNO1 and the FACT complex in the presence or absence of Yap and Tead4.In both cases, Ssrp1 and Spt16 were co-precipitated along with SBNO1 (Fig. 7b), although again precipitation of Tead4 became inefficient in the presence of Yap, as observed in Fig. 6a.When Yap was precipitated, no Ssrp1 or Spt16 was co-purified (IP:α -HA; Fig. 7c), indicating a weak interaction between Yap and the FACT complex.In contrast, Tead4 interacted strongly with Spt16 and Ssrp1, although this interaction became weak when Yap was present (IP:α -FLAG; Fig. 7c).These lines of evidence suggest that the FACT factors make a complex with SBNO1 and Tead4, yet formation of this complex is transient.When co-activators, such as Yap, are recruited to the complex, Spt16 and Ssrp1 are released, which may be a mechanism to control their histone chaperone activity to facilitate transcriptional elongation of target genes. ", "section_name": "Sbno1 physically interacts with FACT (FAcilitates Chromatin Transcription), a histone chaperone for transcription.", "section_num": null }, { "section_content": "Our analysis clarifies a pivotal role of Sbno1 in preimplantation development.The transcriptional level of a key TE determinant, Cdx2, is stimulated by Sbno1 enzymatic activity, along with Yap/Tead4 and Notch/Rbpj transcriptional complexes.Physical interaction between Sbno1, Yap/Tead, Notch/Rbpj and FACT complexes indicates that Sbno1 coordinates association of DNA, transcription factors and histones.These findings describe a critical function of a helicase-related factor on gene transcription during cellular differentiation. During development of mouse preimplantation embryos, genes encoding essential components of the Notch signaling pathway are expressed 47 .Nonetheless, Notch signaling seemed to be dispensable, because maternal/ zygotic knockout embryos of Rbpj, Notch1, or O-fucosyltransferase 1 can implant normally and survive until E9.5 48,49 .Contrary to these observations, Rayon et al., reported Notch/Rbpj activity in TE cells and, more importantly, that expression of Cdx2 in TE cells is regulated by both Notch/Rbpj and Yap/Tead4 through their direct binding to the TEE 7 .Our study also shows that Notch/Rbpj and Yap/Tead4 synergistically activate the Cdx2-TEE in both HEK293 and ES cells, and this synergism was interrupted by the hSBNO1-E437Q mutant, which is deficient in ATPase activity (Fig. 5).The synergistic activation on the Cdx2-TEE was robust when Yap and Tead4 are used in our assay, a clear contrast to Tead4-VP16, which gave only 3-fold activation 7 .This could be a difference between reporter constructs, since we multimerized the Cdx2-TEE elements in our luciferase reporter.As another possibility, an artificial fusion activator Tead4-VP16 could not interact with Yap and/or NICD normally, failing to achieve the physiological activation, which requires Sbno1.Nonetheless, both reports clearly show that the Notch and Yap cascades synergistically activate the Cdx2-TEE, with Sbno1 acting as a signal integrator of these two different cascades. In addition to the TEE region, Tead4 binds to several genomic sites on the Cdx2 locus in blastocysts and trophoblast stem cells 50 , and combination of different regulatory elements might be necessary for the robust Cdx2 expression in the TE 51 .Interestingly, Tead4 knockout embryos can form blastocoel with Cdx2 and other TE gene expression when cultured under a hypoxic condition 52 , and it is intriguing whether regulatory roles of Sbno1 on transcriptional activation and cellular survival might depend on oxygen concentration. Although Sbno1 expression is ubiquitous in preimplantation embryos and at later developmental stages (Supplementary Fig. 1), it can regulate distinct target genes, namely Cdx2, in a temporally and spatially controlled manner via interactions with Yap/Tead4 and Rbpj in the TE of preimplantation embryos.In post-implantation embryos, Sbno1 might control development of the neural tube and presomitic mesoderm, where its expression is evident (Supplementary Fig. 1).Importantly, these two tissues require Notch signaling for their proper development 53,54 .Moreover, Hippo signaling is involved in the control of the size of the neural progenitor pool 55,56 .Although Sbno1 function in the presomitic mesoderm is not known, these data strongly suggest that the same mechanism functions during neural development.Recently, it has been reported that the Notch and Hippo cascades regulate homeostasis of crypts in the intestinal epithelium 57,58 , in which Cdx2 is expressed 59 .Analyses should be expanded to other organs and their cancers, such liver/hepatocarcinoma, colon/colorectal cancers and pancreas/pancreatic cancers, because the Notch and Hippo pathways play critical roles during carcinogenesis [60][61][62] . Sbno1 knockout embryos after E3.5 and Sbno1 knockout ES cells showed remarkable cell death.These results indicate that Sbno1 is an essential factor, not only for the Cdx2 transcriptional regulation but also for the cellular survival in pre-and peri-implantation embryos.FACT complex is known to regulate transcriptional elongation, but also required for DNA repair 63 .Loss of Ssrp1 in mouse embryos causes peri-implantation lethality 64 , and depletion of Ssrp1 or Spt16 in ES cells results in cell death 65 , suggesting that the function of Sbno1 on cellular viability in preimplantation embryo may be associated with functions of the FACT complex. Based on its domain structure, we conclude that Sbno1 belongs to the DExD/H helicase sub-group of the SF2 family 26 .DExD/H helicases are proposed to be ATP-dependent RNA helicases, although several DExD/H helicases are active in other areas of RNA metabolism 23 .More importantly, several members have multiple functions as transcriptional regulators, which are independent of their RNA helicase activity.For example, DDX3, a DEAD-box RNA helicase, is a regulatory subunit of Casein Kinase 1 in the canonical Wnt signaling cascade 66 , highlighting a novel role of a DEAD-box protein as a crucial Wnt signal regulator.In this sense, our data impart a new role to Sbno1 as a transcriptional regulator bridging the Yap/Tead-Notch/Rbpj complexes and the FACT histone chaperone. Previous reports have shown that transcriptional co-activators, such as NcoA6 or the mediator complex, are crucial for transcription of Yap target genes 67,68 , although functional relationship with the histone chaperone is largely unknown.We speculate that binding of Yap and NICD to their binding partners Tead4 and Rbpj might release Sbno1 and the FACT complex to facilitate nucleosome melting, which is essential for efficient transcriptional elongation (Supplementary Fig. 8).In the absence of Sbno1, the FACT complex might loose its access to Cdx2 gene, resulting in a pause of transcriptional elongation that can be found in Yap/Tead targets 67,68 .We do not exclude a possibility that Sbno1 per se might help association of co-activators to the Yap/Tead4 and NICD/Rbpj complexes on Cdx2, since Gal4-Sbno1 acts as a robust transcriptional activator (Fig. 3b).To understand the mechanistic actions of Sbno1 and the FACT complex precisely, future studies must verify whether Sbno1 processes promoter melting and/or interacting domains to the transcriptional co-activators and chromatin remodelers. Although we do not know whether Sbno1 possesses the ATP-dependent helicase activity of the SF2 family members, our analysis has shown that amino acid residue E437 in the DExH box (motif II, ATPase domain) of hSBNO1 is essential for its transcriptional control 30,31 .This is because the E437Q mutant only disrupts the synergism between the Hippo and Notch pathways, but does not inhibit Yap alone or Notch1Δ E alone when used to activate the reporters (Fig. 5c).Our data also suggest that artificial ATP analogs or small chemicals could be designed to abrogate the activity of the DExH box by binding to its pocket.Such chemicals would be antagonistic to the confluence of Yap/Tead and Notch signaling and may, therefore, be good candidates for anti-cancer drugs.It is also of interest that energy stress attenuates the growth-promoting effect of Yap/Tead via AMPK phosphorylation of the Hippo signaling components [69][70][71] .This suggests that ATP-analogs antagonistic to the Yap/Tead and Notch cascade might also be good candidates for anti-cancer drugs. Recently, Hippo signaling was shown to be inhibited by cytoskeletal tension, and high tension sensed by the Ajuba protein, jub, inhibits the Hippo cascade to activate Yorkie-mediated transcription 42 .In addition, Yap was reported to act as a sensor of mechanical cues, such as stiffness of the extracellular matrix 34 and the Notch receptor was shown to be activated by mechanical force via a mechanical allostery of its proteolytic cleavage site 72 .Hence, these two signaling cascades can be activated by mechanical stimuli and/or changes of the physical milieu.Interestingly, nuclear localization of Yap is interrupted when Rho/Rock signaling or myosin II ATPase is inhibited in preimplantation embryos, indicating that cell polarity and contractile force of the cell regulate Hippo signaling 5,[73][74][75] .In preimplantation embryos, stochastic activation of Notch at the morula stage shifted to restricted activation in blastocyst TE cells 7 , but the mechanism of the activation is not understood.Because the TE cells that cover the surface of the preimplantation embryo have a flattened shape whereas the cells inside are round, cell division inside the embryo and subsequent expansion of the blastocoel could apply distinct physical forces to the TE and the cells inside (e.g.stretch vs. compression, respectively).This suggests that this mechanical difference could induce simultaneous activation of the Yap/Tead and Notch/Rbpj complexes, which would be integrated in a synergistic manner by Sbno1.As we have previously shown, physical forces can control gene expression during morphogenesis 76,77 ; therefore, the functional relationship between the physical milieu and gene expression in the formation of the TE should be analyzed further, with particular regard to Yap/Tead, Notch/Rbpj and Sbno1. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Plasmids.δ-crystalline 51 bp minimal promoter (δ51)-LucII and 8xGT-IIc-δ51-LucII were described in refs 29 and 32, respectively.GAL4-UAS-δ51-LucII contains four copies of the Gal4 binding site.Mouse 4xCdx2 TE enhancer (TEE) 47bp-δ51-LucII was constructed by the insertion of four copies of the following fragment containing Tead and Rbpj binding sites (5′ -ggatccTTGACGAATTCCTAAGTCACATATTAATTGTTCCCA CCGAACGCAAAagatct-3′ ) 7 into the δ51-LucII vector.FLAG-mTead4, Gal4-mTead4c and HA-mYap plasmids are described in Nishioka N et al. and Ota M et al. 3,32 .FLAG-mNotch1ΔE (deletion of extracellular domain)-Venus and Myc-mSsrp1 constructs were kindly provided by Dr. Saga 78 and Dr. Murata 79 , respectively.hSBNO1, Myc-mNICD1 (V1744-K2531), FLAG-mRbpj and mSpt16-VSVG expression plasmids were constructed by PCR amplification using appropriate sets of primers. Transfection of DNA and siRNA, and luciferase assay.Twenty-four hours before transfection, 5 × 10 4 293 T or E14Tg2a ES cells/well were seeded in 24-well plates.DNA mixtures of luciferase reporter (0.05 μ g/well), effector (0.1~0.5 μ g/well), CMV-β gal (0.05 μ g/well, as an internal control), and pcDNA3.0(to keep total amounts of transfected DNAs constant) were mixed with three volumes of polyethylenimine or XtremeGENE-HP (Roche) for 293 T cell, or 1.5 ul/well PLUS reagent with 3.75 ul/well Lipofectamine LTX (Invitrogen) for ES cell, and then added to cells.For siRNA experiments, cells were transfected with mixtures of 2 μ l/well XtremeGENE-siRNA (Roche) and 1.5 μ l/well of 20 μ M Sbno1 stealth siRNA (Invitrogen, mixture of #HSS124121, #HSS124122 and #HSS182932) or negative control Low GC duplex (Invitrogen) (the final concentration of siRNA was 50 nM).The medium was changed 24 hours after the siRNA treatment, and then DNA was transfected with three volumes of XtremeGENE-HP (Roche).Transfected cells were cultured for 48 hours and then lysed to measure luciferase activities using a LMAX II luminometer (Molecular Devices).β -Galactosidase activity was measured using an iMark microplate reader (BioRad) to normalize the luciferase activities.All transfections were performed in triplicate, and independently repeated at least three times, which gave reproducible results.For the luciferase assay on mechanically stretched cells, 5 × 10 4 or 1 × 10 5 293 T cells were seeded on fibronectin-coated 2 × 2 cm stretch silicone chambers 24 hours before transfection.DNA mixtures of the luciferase reporter (0.05 μ g/well), effector (0.1 μ g/well), CMV-β gal (0.05 μ g/well, as an internal control) and empty pcDNA3.0(to keep total amounts of transfected DNAs constant) were mixed with three volumes of polyethylenimine and then added to 293 T cells.Transfected cells were cultured for 24 hours.The medium was changed 2 hours before cell stretching.Transfected cells were stretched (1 Hz, 5% stretch, 2 hours) (STB Cell Stretching System, Strex, Osaka, Japan) and then rested in non-stretched conditions for 2 hours.Stretched cells were lysed to measure luciferase activity as described above. Generation of Sbno1 knockout mouse.The Sbno1 knockout mouse line was generated by Ozgene Pty. Ltd. as follows.A loxP-fused region of exon 7 (709 bp) was amplified by PCR from C57BL/6 genomic DNA and subcloned upstream of an FRT-flanked Pgk promoter-Neo resistance gene-polyA (PGK-neo)-loxP cassette.The 5′ -(4,261 bp) and 3′ -homology arms (2,851 bp) were also amplified by PCR from C57BL/6 genomic DNA.These arms were subcloned upstream of the loxP-exon7 fragment and downstream of the PGK-neo cassette, respectively (Supplementary Fig. 2a).This targeting vector was electroporated into Bruce 4 ES cells (derived from C57BL/6).Targeted ES clones were selected by G418 treatment, and analyzed by Southern blot analysis with 5′ and 3′ probes (Supplementary Fig. 2a,b).Correct clones were injected into blastocysts to produce chimeric mice.After breeding the F1 generation, the loxP-flanked exon 7 and PGK-Neo cassette were deleted by crossing with the Oz-Cre mouse strain (Ozgene), which possesses ubiquitous Cre activity.Sbno1 wild type (Wt), floxed (f) and knockout (Δ ) alleles were genotyped by PCR with F; 5′ -AGACTGGTGGTGTGCAGTACC-3′ and R1, 5′ -GAAAGAAGGCTCGGTGGCTAA-3′ or R2, 5′ -CACCACTGCATCAGGGTGAC-3′ primers.F and R1 primers amplify 840 and 250 bp fragments from Wt and knockout alleles, respectively.F and R2 primers amplify 350 and 420 bp fragments from Wt and floxed alleles, respectively (Supplementary Fig. 2a,c).All animal experiments were performed in accordance with institutional guidelines, and full details of the animal experimental protocols were approved and ethical permission was granted by Animal Care Committee of Tohoku University. RNA injection and embryo culture.Zygotes or two-cell stage embryos were collected from oviducts by flushing with M2 medium (Sigma).EGFP-hSBNO1-Wt or -E437Q RNA was synthesized by mMESSAGE mMA-CHINE kit (Thermo) and purified RNA (100 ng/ul) was injected to the zygotes.Embryos were cultured in a drop of KSOM (ARK resource) covered by mineral oil at 37 °C, 5% CO 2 .Pictures were taken using an MZ16 microscope (Leica) and a DFC310FX digital camera (Leica), and fluorescent images were taken by FV1000 (Olympus). ", "section_name": "Methods", "section_num": null }, { "section_content": "Total RNAs of preimplantation embryos were extracted with Trizol reagent (Invitrogen) and cDNAs synthesized using SuperScript III reverse transcriptase (Invitrogen), according to the manufacturer's protocol.PCR was performed with Blend Taq (Toyobo) for 40 cycles of 94 °C for 30 sec, 60 °C for 30 sec, and 72 °C for 30 sec.Primers used are shown in Supplementary Table .Immunohistochemistry, cell death detection and in situ PLA.Preimplantation or cultured embryos were collected and fixed in 4% paraformaldehyde for 30 minutes.After washing with 0.2% lamb serum in PBS (PBSS), embryos were permeabilized by 0.2% Triton-100 in PBS for 30 minutes, and then blocked with 2% lamb serum in PBS.The following primary antibodies were diluted to optimal concentrations in blocking buffer and incubated with embryos overnight at 4 °C: α -Sbno1 (Abcam, #ab122789), α -β -catenin (BD Transduction, #610153), α -phospho-Histone H3 (Cell Signaling, #9701), α -Oct3/4 (MBL, #PM048), α -Nanog (ReproCell, #RCAB002P-F), α -Cdx2 (Biogenex, #MU392A-UC), α -Tbr2 (Eomesodermin) (Abcam, #ab23345), α -Keratin 8 (Developmental Studies Hybridoma Bank, #TROMA-I), α -Yap (Cell Signaling, #4912), α -Tead4 (Abcam, #ab58310), and α -Rbpj (Cell Signaling, #5313).After washing the embryos wish PBSS, secondary antibodies (Alexa Fluor 488, 546 or 594 goat anti-mouse or anti-rabbit IgG, Molecular probes) were diluted 1/1000 in PBSS before incubation for 1 hour.For detection of cell death, embryos were incubated in the TUNEL reaction mixture of the in situ cell death detection kit AP (Roche) for 1 hour at 37 °C.Duolink In situ PLA was performed according to the manufacture's protocol (Sigma).Nuclei were stained with 4′ ,6-diamidino-2-phenylindole in PBS or Topro-3 in 40% glycerol/PBS.Fluorescent images were captured using an FV1000 (Olympus) or TCS-SP5 (Leica) confocal microscope at 3-4-μ m optical sections. Co-immunoprecipitation assay.293 T cells were seeded in a 10-cm dish at a density of 2 × 10 6 cells/dish 24 hours before transfection.Cells were transfected with 3-5 μ g of expression plasmids using XtremeGENE-HP (Roche) according to the manufacturer's protocol.Cells were harvested 48 hours after transfection and lysed in 500 μ l lysis buffer (10 mM HEPES pH 7.6, 250 mM NaCl, 0.1% NP40, 5 mM EDTA and protease inhibitors).After homogenization and brief sonication, the lysates were centrifuged, and the supernatant was subjected to immunoprecipitation with the following antibodies (2 μ g); α -Myc (9E10; Santa Cruz, #sc-40), α -HA (F-7; Santa Cruz, #sc-7392), α -FLAG (DDDDK; MBL, #M185), α -V5 (MBL, #M167-3) or α -GFP (MBL, #598).Twenty microliters of protein-G PLUS-Agarose beads (Santa Cruz, #sc-2002) were added to the lysates, and incubated for 30 minutes at 4 °C.The beads were washed four times with 1 ml lysis buffer, and dissolved in 40 μ l 2xSDS (sodium dodecyl sulfate) sample buffer.Immunoprecipitates were separated by SDS polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membrane.Target proteins were probed with the following primary antibodies: α -Myc (Cell Signaling, #2278), α -HA (Cell Signaling, #3274), α -FLAG (DDDDK; MBL, #PM020), α -V5 (MBL, #PM003), α -GFP (MBL, #598) or α -VSVG (MBL, #563), and then goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody.The membrane was reacted with ECL Prime Western Blotting Detection Reagent (GE Healthcare), and chemiluminescent signals were visualized with ImageQuant LAS 4000 mini (GE Healthcare).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.This work is licensed under a Creative Commons Attribution 4.0 International License.The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/© The Author(s) 2017 ", "section_name": "RT-PCR.", "section_num": null } ]	[ { "section_content": "We thank Dr. T Kihara and Dr. M Murata (Tokyo Univ.) for the Myc-Ssrp1 plasmid construct, Dr. Y Saga (National Institute of Genetics) for the FLAG-Notch1ΔE-Venus plasmid, Dr. Y Matsui (Tohoku Univ.) for E14Tg2a ES cell, Dr. S Kanno (Tohoku Univ.) for Sbno1 structural analysis, Ms. Y Mizuta for help with mouse husbandry, Mr. N Okamura for technical assistance and Ms M Yoshida for secretarial help.This work was supported by Grantsin-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and the Japan Society for the Promotion of Science (JSPS) (to T.O., H.S. and Y.W.), JST-CREST (to T.O.), AMED-CREST (to T.O.) and the program of the Joint Usage/Research Center for Developmental Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University (to H.S. and Y.W.). ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Supplementary information accompanies this paper at http://www.nature.com/srepCompeting Interests: The authors declare no competing financial interests. ", "section_name": "Author Contributions", "section_num": null }, { "section_content": "", "section_name": "Author Contributions", "section_num": null }, { "section_content": "Supplementary information accompanies this paper at http://www.nature.com/srepCompeting Interests: The authors declare no competing financial interests. ", "section_name": "Additional Information", "section_num": null } ]
10.3390/molecules24132367	The Metabolomic Profile of Lymphoma Subtypes: A Pilot Study	<jats:p>Lymphoma defines a group of different diseases. This study examined pre-treatment plasma samples from 66 adult patients (aged 20–74) newly diagnosed with any lymphoma subtype, and 96 frequency matched population controls. We used gas chromatography-mass spectrometry (GC-MS) to compare the metabolic profile by case/control status and across the major lymphoma subtypes. We conducted univariate and multivariate analyses, and partial least square discriminant analysis (PLS-DA). When compared to the controls, statistically validated models were obtained for diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and Hodgkin lymphoma (HL), but not follicular lymphoma (FL). The metabolomic analysis highlighted interesting differences between lymphoma patients and population controls, allowing the discrimination between pathologic and healthy subjects: Important metabolites, such as hypoxanthine and elaidic acid, were more abundant in all lymphoma subtypes. The small sample size of the individual lymphoma subtypes prevented obtaining PLS-DA validated models, although specific peculiar features of each subtype were observed; for instance, fatty acids were most represented in MM and HL patients, while 2-aminoadipic acid, 2-aminoheptanedioic acid, erythritol, and threitol characterized DLBCL and CLL. Metabolomic analysis was able to highlight interesting differences between lymphoma patients and population controls, allowing the discrimination between pathologic and healthy subjects. Further studies are warranted to understand whether the peculiar metabolic patterns observed might serve as early biomarkers of lymphoma.</jats:p>	[ { "section_content": "Lymphomas represent a heterogeneous group of lymphoid malignancies with varied patterns of clinical behavior and responses to treatment.Lymphomas rank the fifth most common cancer in the developed world [1].Prognosis depends on the histologic type, clinical factors, and molecular characteristics.Lymphomas are classified based upon their histological characteristics, and the stage of maturation of the lymphocytes from which they originate [2].B-cell lymphomas are the most frequently represented, and they include diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), multiple myeloma (MM), and other less frequent subtypes. Lymphoma classification keeps evolving thanks to new molecular tools, such as metabolomics.Metabolomics is one of the most recent innovative technologies aiming to understand the metabolic processes within cells, tissues, organs, and organisms.It focuses on the quantitative analysis of a large number of metabolites, representing the end-products of genes, transcripts, and protein functions.The strong interest in metabolomics relates to the fact that even subtle changes in genes, abundance of Molecules 2019, 24, 2367 2 of 11 transcripts, or levels of protein can substantially change the quantity and dynamics of metabolites.Therefore, the analysis of metabolites represents a sensitive measure of the biological status in health or disease [3].Altered metabolic fingerprints of lymphoma patients offer novel opportunities to detect or identify potential risks, and ultimately help achieve the goal of \"personalized medicine\" [4].In this regard, a sizable number of findings have been tested for translational applications, focusing on lymphoma ranging from early detection to therapy prediction and prognosis [5,6]. Recently, a metabolomic approach has been proposed to identify possible biomarkers for characterization and early diagnosis of the different lymphoma subtypes [6].The metabolomic reports published thus far employed different techniques, such as liquid chromatography-mass spectrometry (LC-MS) [7][8][9][10], both gas chromatography-mass spectrometry (GC-MS) and LC-MS [11,12], or nuclear magnetic resonance (NMR) [13][14][15][16], and different bio specimen [7][8][9][10][11][12][13][14][15][16].In this study, a GC-MS technique was used to analyze plasma samples from patients affected by different lymphoma subtypes, and from age (10-year groups) and gender frequency matched population controls.The aim of the study was to identify possible metabolic biomarkers allowing early diagnosis, and possibly differential diagnosis between the subtypes. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "Table 1 shows the gender distribution and mean age of the study population by case-control status.Cases are subdivided by histotypes.We compared the metabolomic profile of patients affected by the five major B-cell lymphoma subtypes to that detected in healthy controls using univariate t-test analysis, multivariate analysis, and partial least square-discriminant analysis (PLS-DA).The following analyses were conducted: Diffuse large B-cell lymphoma (DLBCL) (13 samples vs 42 controls), follicular lymphoma (FL) (8 samples vs 34 controls), chronic lymphocytic leukemia (CLL) (6 samples vs 29 controls), multiple myeloma (MM) (9 samples vs 36 controls), and Hodgkin lymphoma (HL) (10 samples vs 36 controls).Table 2 shows the results of the univariate analysis.The PLS-DA identified four cross-validated models.Table 3 shows the results, and Figure 1 reports the corresponding score plots.The variable importance in projection (VIP) score plots are reported as Supplementary Figures S1-S4.As shown in Table 3, the PLS-DA discriminating ability from the controls was maximum for CLL (Q 2 = 0.734).The comparison between FL and control samples did not result in significant differences in respect to the controls (Q 2 = 0.131), and therefore will not be discussed further.For each comparison, the PLS-DA analysis identified the most important metabolites in the class discrimination.Table 4 shows the relative abundance differences of the most important metabolites for the different comparisons.The PLS-DA identified four cross-validated models.Table 3 shows the results, and Figure 1 reports the corresponding score plots.The variable importance in projection (VIP) score plots are reported as Supplementary Figure S1-S4.As shown in Table 3, the PLS-DA discriminating ability from the controls was maximum for CLL (Q 2 = 0.734).The comparison between FL and control samples did not result in significant differences in respect to the controls (Q 2 = 0.131), and therefore will not be discussed further.For each comparison, the PLS-DA analysis identified the most important metabolites in the class discrimination.Table 4 shows the relative abundance differences of the most important metabolites for the different comparisons.Two metabolites were more abundant in all lymphoma subtypes compared to the controls: Hypoxanthine and elaidic acid.Another interesting feature was the number of metabolites showing a common behavior across the different lymphoma subtypes.In particular, eight metabolites showed a similar upward or downward change in DLBCL and CLL cases compared to the controls, namely 2-aminoadipic acid, 2-aminoheptanedioic acid, 4-hydroxyproline, erythritol, glucoheptonic acid, inositol-like (an inositol isomer other than myo-, scyllo-and chiro-inositol), threitol, and unknown 1910.Among these, 2-aminoadipic acid/2-aminoheptanedioic acid (common name 2-aminopimelic acid), and erythritol/threitol are chemically closely related (Figure 2). a Identified by NIST (matching factor >70%). b Identified by GMD (matching factor >70%). c Identified by in-house library.d Inositol structural isomer other than myo-inositol, chiro-inositol, scyllo-inositol.e Chemical class: AA (Amino acid), HA (Hydroxy acid), A (Acid), FA (Fatty acid), PO (Polyol), Am (Amine), S (Sugar), P (Purine), I (Inorganic). Two metabolites were more abundant in all lymphoma subtypes compared to the controls: Hypoxanthine and elaidic acid.Another interesting feature was the number of metabolites showing a common behavior across the different lymphoma subtypes.In particular, eight metabolites showed a similar upward or downward change in DLBCL and CLL cases compared to the controls, namely 2-aminoadipic acid, 2-aminoheptanedioic acid, 4-hydroxyproline, erythritol, glucoheptonic acid, inositol-like (an inositol isomer other than myo-, scyllo-and chiro-inositol), threitol, and unknown 1910.Among these, 2-aminoadipic acid/2-aminoheptanedioic acid (common name 2-aminopimelic acid), and erythritol/threitol are chemically closely related (Figure 2).In fact, 2-aminoadipic and 2-aminoheptanedioic acids are α-amino bicarboxylic acids differing by only one carbon (i.e., they are homologous), and both were less abundant in DLBCL and CLL patients compared to the controls.Threitol and erythritol are four-carbon polyols differing by the configuration of only one chiral carbon (i.e., they are diastereomers), and both were more abundant in DLBCL and CLL cases compared to the controls.In fact, 2-aminoadipic and 2-aminoheptanedioic acids are α-amino bicarboxylic acids differing by only one carbon (i.e., they are homologous), and both were less abundant in DLBCL and CLL patients compared to the controls.Threitol and erythritol are four-carbon polyols differing by the configuration of only one chiral carbon (i.e., they are diastereomers), and both were more abundant in DLBCL and CLL cases compared to the controls. Eight other metabolites showed similar changes in MM and HL cases compared to the controls, namely cis-aconitic acid, glutamic acid, hippuric acid, myristic acid, oleic acid, palmitoleic acid, and stearic acid.All these metabolites are carboxylic acids; four are fatty acids, two saturated and two unsaturated.All the four fatty acids were more abundant in MM and HL patients compared to the controls. ", "section_name": "Results", "section_num": "2." }, { "section_content": "We analyzed the metabolome of plasma samples from patients of five lymphoma subtypes and healthy controls by untargeted GC-MS.We obtained a significant PLS-DA model for four out of the five major lymphoma subtypes.A common feature of the four significant models was the relative abundance of hypoxanthine and elaidic acid among the patients in respect to the controls.Hypoxanthine is a purine involved in adenine and guanine metabolism and, therefore, in the synthesis of the corresponding nucleosides.In this regard, Yoo found low amounts of hypoxanthine in the urine of non-Hodgkin lymphoma (NHL) patients [7], while plasma levels were elevated in children with acute lymphoblastic leukemia (ALL) or NHL: In these patients, treatment with high-dose methotrexate lowered hypoxanthine levels [17].Serum hypoxanthine levels were also elevated in a heterogeneous group of hemolymphatic malignancies, including acute myeloid leukemia, NHL and CLL [14], and in rectal cancer patients who underwent chemoradiotherapy [18].Uric acid, another purine metabolite, showed higher levels in CLL and MM, and lower in DLBCL and HL when compared to the controls.Uric acid is the end-product of the purine oxidative degradation, deriving from hypoxanthine through xanthine by a NAD-dependent oxidoreductase (https://www.genome.jp/dbget-bin/www_bget?rn: R01768; https://www.genome.jp/dbget-bin/www_bget?rn:R02103). Elaidic acid is the trans isomer of monounsaturated C18 oleic acid, naturally present in ruminant fat, meat, margarine, and baked products [19]; its plasma level has been associated with an increase in total mortality and in cardiovascular mortality [20], and a diet high in trans fatty acids has been associated with an increase in NHL risk [21].Herein, for the first time, we report that elaidic acid plasma level is more elevated in lymphoma patients, likewise in the four subtypes we could investigate, compared to the controls. Other fatty acids, such as myristic, oleic, palmitoleic, and stearic acid were more represented in both MM and HL, and plasma samples from HL patients were also characterized by an increased amount of linoleic and palmitic acid.Dysregulation of fatty acid metabolism in cancer cells is well known [22,23] as it is the potential of fatty acid synthase (FAS) as a drug target; in fact, FAS was expressed above normal in MM [24] and CLL [25,26]. Glycine was more abundant in plasma samples of DLBCL and HL cases compared to the controls.How this observation matches the reported impairment in intracellular glycine transport in DLBCL patients [9] is still unclear.A connection has been suggested between defective intracellular glycine import and increase in tetrahydrofolate-bound one-carbon unit production resulting from conversion from serine to glycine by serine hydroxymethyltransferase (SHMT) [9]; the hypothesis is worth exploring, as previous studies have shown the relevance of one-carbon metabolism and changes in the methylation pattern in the etiology of lymphoma subtypes [27,28]. 2-aminoadipic acid was reported at increased levels in patients with carcinoma of the prostate [29], and it was tentatively proposed as a biomarker of glioblastoma aggressiveness [30].The finding of a higher level of its homologous 2-aminoheptanedioic acid in the cerebrospinal fluid of glioblastoma patients, compared to that of grade I-II and grade III glioma patients [31], and in fecal samples from colorectal cancer patients [32] would support the proposal.On the contrary, levels of the same fatty acids were lower in plasma samples of DLBCL and CLL patients than in controls, and 2-aminoadipic acid was lower in colorectal cancer tissue in respect to the adjacent normal mucosa [33]. Recently, erythritol, a four-carbon bacterial metabolite [34], has been identified as an endogenous human metabolite derived from glucose-6-phosphate in the pentose phosphate pathway (PPP) [35], which would link its production to obesity in young adults.In the present study, erythritol and threitol were more abundant in DLBCL and CLL cases: The links between these metabolites and the PPP would suggest a disorder of the glucose catabolic pathway in these lymphoma subtypes. Consistent with previous reports [14], CLL cases had an elevated level of 2-hydroxybutyric acid, a by-product in the synthesis of glutathione from cystathionine under oxidative stress condition.This four-carbon hydroxy acid was also increased in plasma from hepatocellular carcinoma cases [36], and it was suggested as a potential biomarker of insulin resistance and impaired glucose regulation [37,38]. Our study has several limitations.First, the small sample size did not allow discrimination between the individual major lymphoma subtypes based on their peculiar metabolic features, although we could identify specific metabolic imprints for each in respect to the healthy controls.All patients donated their blood before undergoing treatment, so that we could be reasonably confident that what we observed was in fact a disease effect.Only large-scale follow-up studies in the general population might help in understanding whether the metabolic changes observed could also be predictive of a developing lymphoma in its early stage.Secondly, we performed a large number of comparisons, which might have resulted in a proportionally elevated number of chance findings.However, we corrected p-values using the false discovery rate technique, and we interpreted our results consequently, based also on their consistency with previous literature reports. In spite of such limitations, we think our findings warrant replication in larger pooled analyses. ", "section_name": "Discussion", "section_num": "3." }, { "section_content": "", "section_name": "Materials and Methods", "section_num": "4." }, { "section_content": "During 2012-16, we recruited incident adult patients (aged 20-74) with a first diagnosis of lymphoma at the hematology unit of the A. Businco Hospital in Cagliari-the main referral center for oncohematology in southern Sardinia, Italy-to participate in a case-control study on gene-environment interactions in the etiology of lymphoma.The pathologists collaborating to the study reviewed the clinical diagnosis of lymphoma using the 2008 World Health Organization (WHO) classification of lymphoma.All lymphoma subtypes, including B-cell and T-cell lymphomas, and Hodgkin lymphoma were included.Controls were a random sample of the resident population in southern Sardinia, the referral area of the hematology department of the oncology hospital.Controls were frequency matched to the cases by gender, 10-year age group, and local health unit of residence.Patients affected by infectious diseases and suffering from immune system disorders were ineligible to serve as controls. Following the Helsinki protocol, all study subjects provided written consent to the use of their biological samples before participation, in which they acknowledged that their samples would have been fully anonymized, and their identity could not be identified via the papers or in the databases.The study protocol included an in-person interview, conducted by trained interviewers at the hospital or the residence home; at the end of the interview, subjects were requested to donate a 40 mL blood sample to investigate genetic and epigenetic determinants of disease.Overall, samples were available for 196 cases and 151 controls; after storing plasma samples for the main analyses originally planned, aliquots for 66 cases and 96 controls remained available to study the metabolic profile of lymphoma subtypes, with reference to the controls.After collection, blood samples were centrifuged, and plasma samples were aliquoted and stored at -80 • C until metabolomic analysis. ", "section_name": "Study Population", "section_num": "4.1." }, { "section_content": "The analytical method has been described elsewhere [39], but it was slightly modified for the purposes of the present study.In brief, 400 µL plasma aliquots were treated with 1200 µL of cold methanol in 2 mL Eppendorf tubes, vortex mixed, and centrifuged 10 min at 14,000 rpm (16.9 G). 400 µL of the upper phase were transferred in glass vials (1.5 mL) and evaporated to dryness overnight in an Eppendorf vacuum centrifuge.50 µL of a 0.24 M (20 mg/mL) solution of methoxylamine hydrochloride in pyridine was added to each vial, samples were vortex mixed, and left to react for 17 h at room temperature in the dark.Then 50 µL of MSTFA (N-Methyl-N-trimethyl-silyltrifluoroacetamide) were added and left to react for 1 h at room temperature.Samples were subsequently diluted with hexane (100 µL), with tetracosane (0.01 mg/mL) as the internal standard, just before GC-MS analysis.Analyses were performed on an Agilent 5977B GC/MS interfaced to the GC 7890B (Agilent Technologies, Palo Alto, CA, USA), equipped with a DB-5ms column (Agilent J&W Scientific, Folsom, CA, USA).Injector temperature was 230 • C, detector temperature 280 • C, helium carrier gas flow rate of 1 mL/min.GC oven temperature program was the following: 90 • C initial temperature, 1 min hold time, increasing 10 • C/min to a final temperature of 270 • C, 7 min hold time.Samples (1 µL) were injected in split (1:4) mode.After a solvent delay of 3 min, mass spectra were acquired in full scan mode using 2.28 scans/s with a mass range of 50-700 Amu.Each acquired chromatogram was analyzed by means of the free software AMDIS (Automated Mass spectral Deconvolution and Identification System) (http://chemdata.nist.gov/mass-spc/amdis),that identifies each chromatographic peak by comparison of the relative mass spectra and the retention times with those stored in an in-house library comprising 255 metabolites.Other metabolites were identified using NIST08 (National Institute of Standards and Technology's mass spectral database) and the Golm Metabolome Database (GMD) (http://gmd.mpimp-golm.mpg.de/).Through this approach, 108 compounds were detected and quantified: 97 were accurately identified and 11 compounds were not identified and were defined as unknown. ", "section_name": "Samples Preparation and GC-MS Analysis", "section_num": "4.2." }, { "section_content": "For the metabolomic analysis, the AMDIS data matrix including 108 metabolites was processed with the integrated web-based platform MetaboAnalyst 4.0 [http://www.metaboanalyst.ca/][40].Missing values were replaced with half of the minimum positive values in the original data, and after normalization by sum, data were log transformed and categorized using Pareto scaling for the purposes of analysis, including univariate analysis, partial least square discriminant analysis (PLS-DA), and its associated variable importance in projection (VIP) score.PLS-DA models were tested with the leave-one-out cross validation (LOOCV) method for the evaluation of statistical parameters (correlation coefficient R 2 , cross validation coefficient Q 2 ) [41], which allowed us to determine the optimal number of components for the model description. ", "section_name": "Statistical Analysis", "section_num": "4.3." } ]	[ { "section_content": "The authors are thankful to the patients and the population controls who participated in the study. ", "section_name": "Acknowledgments:", "section_num": null }, { "section_content": "The following are available online. The authors declare no conflict of interest. ", "section_name": "Supplementary Materials:", "section_num": null }, { "section_content": "The following are available online. ", "section_name": "Supplementary Materials:", "section_num": null }, { "section_content": "", "section_name": "Author", "section_num": null }, { "section_content": "The authors declare no conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.1590/s1679-45082011ao2027	Correlation between flow cytometry and histologic findings: ten year experience in the investigation of lymphoproliferative diseases	<jats:p>Objective: To demonstrate the advantages of correlating flow cytometry immunophenotyping with the pathology/ immunohistochemistry of lymph nodes or nodules in the diagnosis of lymphoproliferative diseases. Methods: A retrospective study was carried out of 157 biopsy or fine-needle aspiration lymph nodes/ nodule specimens taken from 142 patients, from 1999 and 2009. The specimens were simultaneously studied with fow cytometry and pathology at Hospital Israelita Albert Einstein. The specimens were prepared in hematoxylin/eosin, Giemsa, or monoclonal antibody stained slides for detecting specific antibodies for the purposes of pathology/immunohistochemical analysis. The samples were hemolyzed and marked with different monoclonal antibody panels for different antigens in fow cytometry immunophenotyping. Results: The diagnostic results of pathology/immunohistochemical studies and flow cytometry immunophenotyping agreed in 115 patients (81%), corresponding to 127 specimens, as follows according to the pathologic diagnosis: 63 patients with non-Hodgkin's B-cell lymphoma; 26 patients with reactive lymphoid hyperplasia; 5 patients with non-Hodgkin's T-cell lymphoma; 4 patients with atypical lymphoid proliferation; 5 patients with a chronic granulomatous inflammatory process; 5 patients with a non-hematologic diagnosis; 2 patients with granulocytic sarcoma; 2 patients with thymoma; 1 patient with byphenotypic leukemia; 1 patient with kappa plasmocytoma; 1 patient with Hodgkin's lymphoma. Subtypes of lymphomas could be classified by associating the two techniques: 19 patients with follicular lymphoma; 15 patients with diffuse large B-cell lymphoma; 7 patients with small lymphocytic B-cell lymphoma/chronic lymphocytic leukemia; 3 patients with mantle cell lymphoma; 1 patient with Burkitt's lymphoma; 1 patient with MALT type lymphoma; 1 patient with post-transplant lymphoproliferative disease; 2 patients with high grade non-Hodgkin's B-cell lymphoma; 1 patient with low grade non-Hodgkin's B-cell lymphoma not otherwise specified; 1 patient with Hodgkin's lymphoma; and 12 patients with B-cell non-Hodgkin's lymphoma not otherwise specified. Conclusion: Flow cytometry adds to the results of morphologic and immunohistochemical studies, facilitating a rapid and accurate diagnosis of lymphoproliferative diseases.</jats:p>	[ { "section_content": "Bezerra AMPS, Pasqualin C, Guerra JCC, Colombini MP, Velloso EDRP, Silveira PAA, Mangueira CLP, Kanayama RH, Nozawa ST, Correia R, Apelle AC, Pereira WO, Garcia RG, Bacal NS Israelita Albert Einstein, em São Paulo.Para a análise na anatomia patológica, as amostras foram preparadas em lâminas e coradas com hematoxilina-eosina, Giemsa, ou marcadas com anticorpos monoclonais para detecção de antígenos específicos.Para a análise por imunofenotipagem por citometria de fluxo, as amostras foram hemolisadas e marcadas com diferentes painéis de anticorpos monoclonais para detecção dos diferentes antígenos.resultados: Foram concordantes os diagnósticos entre a anatomopatológico e imunofenotipagem por citometria de fluxo em 115 (81%) pacientes, o que correspondeu a 127 amostras distribuídas da seguinte forma, conforme o diagnóstico anatomopatológico: 63 pacientes com linfoma não Hodgkin de células B; 26 pacientes com hiperplasia linfoide reacional; 5 pacientes com linfoma não Hodgkin de células T; 4 pacientes com proliferação linfoide atípica; 5 pacientes com processo inflamatório crônico granulomatoso; 5 pacientes com diagnósticos não hematológicos; 2 pacientes com sarcoma granulocítico; 2 pacientes com timoma; 1 paciente com leucemia bifenotípica; 1 paciente com plasmocitoma Kappa; e 1 paciente com linfoma de Hodgkin.A correlação entre os resultados das duas técnicas permitiu a classificação dos subtipos de linfomas da seguinte forma: 19 pacientes com linfoma folicular; 15 pacientes com linfoma difuso de grandes células B; 7 pacientes com linfoma linfocítico de pequenas células B/leucemia linfocítica crônica; 3 pacientes com linfoma de células do manto; 1 paciente com linfoma de Burkitt; 1 paciente com linfoma do tipo MALT (tecido linfoide associado à mucosa); 1 paciente com doença linfoproliferativa pós-transplante; 2 pacientes com linfoma não Hodgkin de células B de alto grau; 1 paciente com linfoma não Hodgkin de células B de baixo grau; 1 paciente linfoma de Hodgkin; e 12 pacientes com linfoma não Hodgkin de células B, sem outra especificação.conclusão: A imunofenotipagem por citometria de fluxo complementa os achados do estudo anatomopatológico/ imunoistoquímico, permitindo um diagnóstico hematopatológico rápido e preciso das doenças linfoproliferativas. Descritores: Linfoma; Transtornos linfoproliferativos/diagnóstico; Imunofenotipagem; Citometria de fluxo; Imunoistoquímica intrODUctiOn Lymph node neoplasms often affect lymph nodes, the spleen, mucosa-associated lymphoid tissues, the skin, or non-lymphoid solid organs, resulting in tumors and enlarged organs.The diagnosis of lymphoproliferative diseases is made by pathology and immunohistochemistry of lymph nodes, which are the gold standard.These approaches demonstrate tissue structure and the classification of the type of lymphoma.These procedures, however, are time consuming and invasive; furthermore, some tissues are difficult to access. Fine needle aspiration biopsy (FNAB) is often the first investigation (screening) for the differential diagnosis among benign/reactional and malignant tumors in cases of enlarged lymph nodes.It is a fast, simple, safe, and only slightly invasive technique for gathering well-preserved cells for studies. FNAB may be used not only in tissue biopsies for pathology, but also as a technique for obtaining tissue specimens for other studies, including flow cytometry immunophenotyping, (1,2) because of its high positive predictive value.Several studies have been proposed for establishing the reliability of FNAB in the diagnosis of lymphoproliferative diseases (3)(4)(5)(6)(7)(8)(9) (Chart 1). Immunological marker analysis of lymph nodes based on flow cytometry immunophenotyping has progressed rapidly in the last few decades, going from restricted use in research to routine use in laboratory diagnoses.This has become possible mainly by the wide availability of flow cytometers and the significant amount of monoclonal antibodies for rapidly detecting membrane and intracellular antigens in different cell suspensions (10,11) . The immunophenotyping study of lymphoproliferative processes is used for distinguishing benign reactions and malignancies; it identifies monoclonality -mainly of B cellswhere there is restriction of one of the light immunoglobulin chains (Figure 1).Besides diagnosis, flow cytometry immunophenotyping is also applied for classifying the types and subtypes of lymphoproliferative diseases (12)(13)(14) . The current classification of lymphoproliferative diseases (World Health Organization) (14) emphasizes histologic, clinical, cytologic, immunophenotypic, and genotypic aspects for diagnosing and defining the prognosis of lymphoproliferative diseases.Thus, new highly specific markers are described on an ongoing basis to improve the diagnosis and to yield information about the prognosis of these diseases; monoclonal antibody panels may include these new markers depending of the needs of clinical investigation (15)(16)(17) . High quality smears are useful, since a differential diagnosis may be made based on the nature of cells (monomorphic; polymorphic; small, medium or large the diagnosis.A κkappa/λlambda proportion below 0.5 or over 3.0 suggests the presence of a clonal B cell population in peripheral blood, bone marrow, lymph nodes, the spleen, or other tissues with larger numbers of mature B lymphocytes (18) . ", "section_name": "", "section_num": "" }, { "section_content": "The purpose of this study was to demonstrate the advantages of correlating flow cytometry immunophenotyping and pathology/immunohistochemistry of enlarged lymph nodes and/or nodules in the diagnosis of lymphoproliferative diseases. ", "section_name": "OBJectiVe", "section_num": null }, { "section_content": "A retrospective study was made of 157 biopsy or fine needle aspiration specimens of lymph nodes or nodules obtained from 142 patients from 1999 to 2009; the specimens were sent simultaneously to the Flow Cytometry Unit and the Pathology Unit of the Israelita Albert Einstein Hospital, São Paulo, SP. ", "section_name": "MetHODS", "section_num": null }, { "section_content": "Biopsies or FNAB of lymph nodes or nodules were done in all patients for histologic and immunohistochemical diagnosis.The hematologic and laboratory routine practices were not altered or interfered with for this study. Pathologists at our hospital usually have three moments to evaluate fresh tissue samples: during ultrasonography for FNABs, during computed tomography for guided needle biopsies, and during intraoperative freeze sections in the surgical theater. Pathologists promptly examined FNABs specimens of lymph node/masses to establish cell representativeness; before tissue fixation of the specimens in 95% alcohol), the material was transferred to a tube containing ethylenediaminetetraacetic acid (EDTA) and 2 mL of a RPMI culture medium (RPMI 1640, developed at the Roswell Park Memorial Institute).Flow cytometry immunophenotyping took place within 6 hours of obtaining the specimen, which precluded the need for fixation. For the biopsies, pathologists selected representative samples of fresh specimens for tissue fixation (10% formaldehyde).The specimens were then transferred to a tube containing EDTA, and flow cytometry immunophenotyping was done similarly to the FNAB cases. In the pathology laboratory, smears and cell centrifugates of FNAB specimens were routinely prepared sized).Attention should be given to significantly hemodiluted materials, because proliferative cases may be mistakenly diagnosed as reactional. The immediate morphological evaluation of specimens after fine needle aspiration may lead to a second FNAB or to a lymph node biopsy to obtain a specimen with more adequate cellularity (1,6,16,18,19) . B cells comprise about 40% and T cells comprise about 55% of normal lymph nodes.The subtype CD4 predominates among CD3+ cells, and the CD4/CD8 ratio is over 4. The frequency of natural killer cells in normal lymph nodes is very low (about 1%).On the other hand, the tonsils are lymphoid organs in which B cells (CD19+) predominate; the remaining cells are CD3+ with a predominance of the CD4 subtype, as in lymph nodes (20,21) . B cell lymphomas are the majority among non-Hodgkin lymphomas; in such cases, establishing cell clonicity -by restriction of one of the κ (kappa) or λ (lambda) light chains -is generally the key to define Source: Laboratory of Special Techniques, Flow Cytometry Unit, Hospital Israelita Albert Einstein Bezerra AMPS, Pasqualin C, Guerra JCC, Colombini MP, Velloso EDRP, Silveira PAA, Mangueira CLP, Kanayama RH, Nozawa ST, Correia R, Apelle AC, Pereira WO, Garcia RG, Bacal NS for cytology; the Papanicolaou and Giemsa fixation were used.Biopsies went through routine histologic preparation and the slides were hematoxylin-eosin stained. Immunohistochemistry consisted of placing the specimens on glass slides previously prepared with a poly-D-Lysine adhesive (Sigma, St. Louis, MO, US, code P7886) and kept in an oven at 60 o C for 4 hours.Deparaffining was done with repeated xylol baths, absolute ethyl alcohol, and washing with a buffered saline solution, a phosphate buffer solution (PBS), and blockage of endogenous activity with a 3% H2O2 solution.Antigenic recovery was attained by heat or the enzyme method.After recovery of the epitopes, the slides were incubated with the primary antibodies for 12 to 18 hours at 4 o C at appropriate dilutions for each antibody.The slides were then washed again with PBS and incubated for 60 minutes with the respective secondary antibodies.Polymer detection systems were then applied.The slides were processed by a treatment with 3,3'-diaminobenzidine (DAB, Sigma, St. Louis, MO, US, code.D5637), H2O2 (final concentration = 0.2%), Mayer hematoxylin counterstained, and mounted with histologic resin.Pathologists evaluated all assays with common microscopy; the immunohistochemical reaction controls were positive. An initial panel consisted of the following primary antibodies: CD20 (clone L26), CD3 (polyclonal), CD10 (clone 56C6), Bcl-2 (clone Bcl-2-100), Bcl-6 (clone lymph node22), CD5 (clone RTB-CD5), CD23 (clone 1B12), cycline D1 (clone SP4), Ki-67 (clone SP6), CD30 (clone Ber-H2), Epstein Barr virus (EBV -clone CS.1-4), and CD15 (clone BY87).This panel was increased by adding the following antibodies, as needed: CD138 (clone MI15), KappaΚ (polyclonal), LambdaΚ (polyclonal), CD4 (clone 1F6), CD8 (clone C8/144B), CD43 (clone DF-T1), CD56 (clone 123C3), myeloperoxidase (polyclonal), granzyme B (polyclonal), TIA-1 (clone C-20), multiple myeloma-1/ interferon regulatory factor-4 (MUM1/IRF4 -clone MUM 1P), and terminal deoxynucleotidyl transferase (TdT -polyclonal), cytokeratins (clone AE1/AE3), Melan A (clone M27C10), protein S-100 (polyclonal), and HMB45 (clone HMB45). Pathologists carried out the final histologic evaluation under a common light microscope, based on the 2001 and 2008 tumor classification systems of the World Health Organization (WHO), as recommended in the literature (14) . ", "section_name": "Pathology/immunohistochemistry and cytology", "section_num": null }, { "section_content": "FNAB samples were placed in a collecting medium (Vitrocell), and lymph node/mass samples were imbibed in a saline solution or a collecting medium (RPMI, Vitrocell).Cells were first counted in a Neubauer chamber.The slides were prepared in Cytospin and colored with a Rosenfeld dye for cytomorphology.The 7-AAD cell viability assay was used.After the morphologic analysis, the specimens were pipette in 12 x 75 mm tubes depending on the sample volume and the number of cells.The specimens were then PBS (phosphate buffer) washed before marking with monoclonal antibodies.These were obtained from several manufacturers: Beckman Coulter (BC), Becton Dickinson (BD), IQ Products (IQP), Immunotech (IM). Basic screening of the phenotypic profile of the specimens consisted of applying a panel with the following antibodies: anti-CD2(BC), anti-CD3(BC), anti-CD4(BC), anti-CD8(BC), anti-CD14(BC), anti-CD15(IM), anti-CD19(BC), anti-CD30(IM), anti-CD34(IM), anti-CD45(IM), anti-Kappa (Dako), and anti-Lambda (Dako) (22) . If clonality was present, a complete antibody panel was used, including a panel for the B cell proliferative disease: CD2(BC), CD3(BC), CD5(IM), CD7(BC), CD10(IM), CD11c(IM), CD20(IM), CD22(IM), CD23(Dako), CD25(BC), CD38(IM), CD79b(IM), CD103(IQP), FMC-7(IM), HLA-DR(IM), IgM(Dako), IgD(Dako), and IgG(Dako). Other panels were used, as follows: -panel for T cell lymphoproliferative disease: CD1a(IM), CD2(BC), CD3(BC), CD5(IM), CD7(BC), CD10(IM), CD20(IM), CD38(IM), CD56((IM), TCR Alfa/Beta(IM), TCR Gamma/Delta(IM); -panel for multiple myeloma and associated diseases: CD19(PC5), CD20(CD5), CD33(IM), CD38(IM), CD45(IM), CD56(IM), CD117(IM), HLA-DR(IM), and intracytoplasmatic markers for Kappa, Lambda, IgM, IgG, IgD, and IgA. After marking with monoclonal antibodies, cells were incubated during 15 minutes at room temperature and away from light.A hemolytic buffer (ammonium chloride) was applied during 15 minutes at room temperature for lysis of red blood cells. Specimens were washed three times with PBS and fetal bovine serum, and incubated during one hour in a water bath at 37 o C for surface marking into light and heavy chain immunoglobulins.The IntraPrep kit (Beckman Coulter) was used for intracytoplasmatic marking.Data gathering and analysis was done in an EPICS XL-MCL and FC-500 (Beckman Coulter) flow cytometer.Analyses were interpreted based on the resulting histograms together with cytomorphology of the specimens, according to the tumor classification system of the WHO( 2001 and 2008) or others recommended previously in the literature (14) . The combined results of lymph node analysis by FNAB cytology and pathology/immunohistochemistry were compared with the results of flow cytometry immunophenotyping.Sensitivity and specificity were used as parameters for assessing the performance of flow cytometry immunophenotyping relative to pathology (the gold standard). Sensitivity was calculated to assess the proportion of diseases subjects that tested positive, and specificity was calculated to assess the proportion of disease-free subjects that tested negative.The positive predictive value was calculated to assess the probability of a subject having the disease when tested positive, and the negative predictive values was calculated to assess the probability of a subject not having the disease when tested negative. ", "section_name": "Flow cytometry immunophenotyping", "section_num": null }, { "section_content": "There were 157 specimens of 142 patients during the period from 1999 to 2009, of which 75 were male and 67 were female; the mean age was 55 years (ranging from 4 to 92 years). The procedures for obtaining the specimens consisted of biopsies in 119 patients, FNAB in 16 patients, and FNAB followed by biopsy in 7 patients. The sites for 145 lymph node specimens were the neck, inguinal, axillary, mediastinal, peripancreatic, paraaortic, and juxtacarotid regions; the sites of 12 tumor mass samples were the spleen, kidney, small intestine, lung, ischium, parotid, scalp, and nasopharynx.There were more than one specimens in 12 patients because of different procedures (for instance, FNAB specimens followed by biopsy specimens), different sites obtained at the same time, different years in a single patient, or duplication of specimens (Table 1). To investigate the efficacy of associating flow cytometry immunophenotyping with pathology for accurate diagnoses, we assessed the agreement percentage between the two techniques for each disease group in the study (Figure 2).The 142 study patients were classified according to the diagnosis of the disease (Table 1).The bars show the percentage of agreement in diagnosis by each technique (Figure 2). ", "section_name": "reSUltS", "section_num": null }, { "section_content": "Interestingly, agreement was above 80% in 9 of 11 disease groups; it was above 70% in one group.Only the diagnosis of Hodgkin's lymphoma (LH) was mostly discordant, which has been predicted in the literature, as will be discussed below. Of 73 patients diagnosed with B cell non-Hodgkin's lymphoma in pathology (51.4% of patients in this study), the agreement with flow cytometry immunophenotyping was 86.3%.There was poor diagnostic agreement in seven patients with T cell non-Hodgkin's lymphoma (4.9% of patients in this B-NHL: B-cell non-Hodgkin lymphoma (n = 73); reactional: reactional lymphoid hyperplasia (n = 26); T-NHL: T-cell non-Hodgkin lymphoma (n = 7); Atypical lymphoid proliferation (n = 5); Chronic granulomatous inflammation (n = 5); Non-hematological: includes carcinomas, adenocarcinomas and melanoma (n = 5); Biphenotypic: biphenotypic leukemia (n=1); HL: Hodgkin's lymphoma (n = 15); Granulocytic sarcoma (n = 2); Thymoma (n = 2); Plasmocytoma (n = 1).Bezerra AMPS, Pasqualin C, Guerra JCC, Colombini MP, Velloso EDRP, Silveira PAA, Mangueira CLP, Kanayama RH, Nozawa ST, Correia R, Apelle AC, Pereira WO, Garcia RG, Bacal NS study).The agreement between pathology and flow cytometry immunophenotyping was 80% in the five patients with atypical lymphoid proliferation.Flow cytometry immunophenotyping was completely effective in the diagnosis of reactional hyperplasia, granulomatous inflammation, non-hematologic cancer, granulocytic sarcoma, thymoma, and individual cases of biphenotypic leukemia and plasmacytoma (100% agreement with pathology). Figure 3 shows the diagnoses of patients according to pathology. -post-transplant lymphoproliferative disease (PTLD): 1 patient; -high grade B cell non-Hodgkin's lymphoma: 2 patients; -low grade B cell non-Hodgkin's lymphoma: 1 patient; -B cell non-Hodgkin's lymphoma, not otherwise specified: 12 patients; -Hodgkin's lymphoma: 1 patient (Figure 5A and5B).The distribution according to subtypes in 63 concordant cases of non-Hodgkin's lymphoma was as follows: -follicular lymphoma (LF): 19 patients (Figure 4A and 4B); -large B cell diffuse lymphoma: 15 patients; -lymphocytic lymphoma/chronic lymphocytic leukemia (LLC/ LL): 7 patients; -mantle cell lymphoma: 3 patients; -Burkitt's lymphoma: 1 patient; -MALT lymphoma (mucosa-associated lymphoid tissue): 1 patient; The following subtypes were found when the diagnoses of non-Hodgkin's lymphoma made with flow cytometry immunophenotyping and pathology did not agree: -B cell lymphoma rich in T cells and histiocytes: 2 patients; -large B cell diffuse lymphoma: 6 patients; -precursor B cell lymphoblastic lymphoma / lymphoblastic leukemia: 1 patient; -marginal zone (MALT) B cell non-Hodgkin's lymphoma with focal involvement of lymph nodes: 1 patient The sensitivity test value for flow cytometry immunophenotyping compared to pathology (gold standard) was 0.77; the specifi city value was 0.97.The positive predictive value was 0.77, and the negative predictive value was 1.00. ", "section_name": "Pathological diagnosis number of patients", "section_num": null }, { "section_content": "The traditional technique of choice for diagnosing lymph node diseases has been histopathology of paraffi n-included tissues.Immunohistochemistry is an important tool for analyzing biopsies of lymph nodes and other tissues; cell morphology and tissues architecture are preserved, and immunophenotypic analysis of histological sections are possible (23,24) .Detecting specifi c antigens in lymphoid cells is fundamental for classifying tumors, assessing the outcome, and identifying specifi c targeted therapy (13,14) .However, pathology of immunohistochemical results in routine laboratory work has its limits: the analysis may be subjective, reproducibility is limited, and the process is time-consuming.Inter-and intra-observer variability is high because so many factors may interfere with the processing of specimens and interpretation of results.Thus, lack of consensus in quantifying antigen expression and defi ning positive and negative results in poor reproducibility.Cytomorphological assays and fl ow cytometry immunophenotyping overcome some of these hurdles by providing faster diagnoses, quantitative and qualitative analysis of cell antigens, and multiparametric analyses (2,7,17,18) .However, fl ow cytometry also has its limits: variability in antigenic signature expression; loss of cells during the pre-analytical process; specimen preparation issues; work with fresh specimens; and availability of suffi cient neoplastic cells.Large cell lymphoma cells may be lost in the preparation process because of cell frailty.According to the literature, a negative result does not exclude malignancy (11,18,19,23) . FNAB is a minimally invasive procedure for which cytomorphological analysis combined with fl ow cytometry immunophenotyping are important tools, which are able to rapidly differentiate lymphoproliferative diseases from reactional lymphoid hyperplasia in most cases of enlarged lymph nodes (20,23,25) . In our study, the diagnosis by fl ow cytometry immunophenotyping and pathology differed in 27 cases; this occurred more often when diagnosing Hodgkin's lymphoma in 14 patients out of 15 patients with positive pathology for this disease (51.85% of the total number of discordant cases), in 17 specimens (56.7%). Although fl ow cytometry is useful for diagnosing several hematopoetic neoplasms, and may often detect small cell populations (< 0.01% of leukocytes), it is a limited technique for the diagnosis of Hodgkin's lymphoma involving lymph nodes.Many studies on fl ow cytometry immunophenotyping in Hodgkin's lymphoma have shown changes in reactional lymphocytes, such as the CD4/CD8 ratio in T cells; however, this technique fails to detect Reed Sternberg cells, especially because of their large volume (26) .In 2009, Wood described a highly sensitive and specifi c technique based on fl ow cytometry using nine colors and three lasers for diagnosing classic Hodgkin's lymphoma (27) . Flow cytometry failed to diagnose non-Hodgkin's B lymphoma in ten patients of our sample.In these cases, there was partial distribution of anomalous cells in lymphoid tissues in two patients, which affected neoplastic cell representation.In two cases, the fi nal diagnosis was large B cell diffuse lymphoma rich in T cells/histiocytes, where those few detected neoplastic cells are spread out in a rich background of T lymphocytes and histiocytes; furthermore, the cells are large and more fragile compared to other lymphocytes.Thus, they are not well represented, which may mask flow cytometry analysis.Meda et al. (28) and Verstovsek et al . (1) also reported this finding.They are often wrongly characterized as a polyclonal population because of significant contamination by residual normal cells.Therefore, a flow cytometry immunophenotyping result showing no evidence of malignancy did not exclude a cancer; in these situations, a detailed cytomorphological exam is required, as demonstrated in the (9,29) .According to Meda et al. (28) and Zardawi et al., (18) if κ and λ light chains are not restricted, further evidence of clonal proliferation may be investigated, such as major antigen proliferation (CD19, CD20) in specific tissues (over 85%), CD10 ≥> 18% or CD20+CD5+ ≥> 35%. Among other studies, Martins et al. (7) conducted a retrospective study of 627 lymph node FNAB specimens and underlined the importance of cytomorphological analysis for the diagnosis of large cell non-Hodgkin's lymphoma. In three of our patients, the specimens consisted of necrotic material, which was frail or sparse for flow cytometry immunophenotyping; the specimen with sparse material was obtained in a bone biopsy, and was not representative of the neoplasm.Representativeness of malignancy was lost in three patients, and flow cytometry immunophenotyping diagnosis was not possible (in one, the PCR technique for B clonality was used to supplement the study); furthermore no distinct representative IgH loci monoclonal rearrangement band was found, which demonstrated the paucity of cells in the specimen.The presence of necrosis, accelerated tumor growth, and bone, affected pre-analytical processing, thereby interfering with cell viability for analysis in the cytometer; this again has been reported in the literature (19,23) . In T cell lymphomas, cell clonality may only be characterized if antigenic expression of a T lineage marker is absent.A demonstration of T clonality may be done using PCR, the Southern Blot molecular biology technique, or flow cytometry for clonality detection in the Vβ family with simultaneous analysis of more than 20 monoclonal antibodies -a technique that is not available in Brazil. In our study, flow cytometry was more specific than sensitive in diagnostic agreement, a result of poor agreement in patients with Hodgkin's lymphoma, (14,15) and in ten patients with large cell diffuse lymphoma, as described previously (10) .These factors also affected the global positive predictive value. Immunohistochemistry is an important tool in biopsies of lymph nodes and other tissues; it is possible to analyze the immunophenotype of histological sections where identification of the tissue architecture is also possible (30) .For example, identifying CD5 antigen expression in some lymphomas, such as between the small cell lymphocytic lymphoma/chronic lymphocytic leukemia (LL/CLL) and the mantle cell lymphoma (ML), which are two types of lymphoproliferative diseases that progress differently, thereby requiring a correct definition.These cases require investigating and charactering the D1 cyclin marker, which is found in 70 to 80% of mantle cell lymphoma cases (15) -a technique that is done successfully only with immunohistochemistry. Detection is also possible of the typical t(11:14) of the mantle cell lymphoma in classic cytogenetics, fluorescent in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR). In mature B cell lymphomas, the main differential diagnosis to be made among those that are positive for the CD10 marker is between the follicular lymphoma, the large cell diffuse lymphoma, and Burkitt's lymphoma.Demonstrating histologically the Bcl-2 marker helps identify neoplastic follicles in follicular lymphomas and differentiate them from reactional follicles in follicular lymphoid hyperplasia.Identifying Bcl-2 is difficult in flow cytometry, which however may differentiate follicular lymphomas from lymphoid hyperplasia by testing clonality in the κ and λ ratio (16,21,31) . Diagnostic centers that currently provide these technologies as supplementary diagnostic tools reduce the limitations of each method, add speed, and further choices for staging and defining the most appropriate treatment. ", "section_name": "DiScUSSiOn", "section_num": null }, { "section_content": "We have been able to show that in several situations, for many hematologic diseases, flow cytometry associated with cytomorphology and immunohistochemistry made it possible to diagnose and differentiate reactional processes from neoplasms, and to subclassify lymphoproliferative diseases. In our experience with the majority of suspected cases of lymphoproliferative diseases, flow cytometry data supplemented the findings of cytomorphology, immunohistochemistry (FNABs) and biopsy specimens. ", "section_name": "cOnclUSiOn", "section_num": null } ]	[ { "section_content": "Conflict of interest: none einstein.2011; 9(2 Pt 1):151-9 ", "section_name": "", "section_num": "" } ]
10.1371/journal.pone.0029094	MicroRNA-449a Overexpression, Reduced NOTCH1 Signals and Scarce Goblet Cells Characterize the Small Intestine of Celiac Patients	MiRNAs play a relevant role in regulating gene expression in a variety of physiological and pathological conditions including autoimmune disorders. MiRNAs are also important in the differentiation and function of the mouse intestinal epithelium. Our study was aimed to look for miRNA-based modulation of gene expression in celiac small intestine, and particularly for genes involved in cell intestinal differentiation/proliferation mechanisms. A cohort of 40 children (20 with active CD, 9 on a gluten-free diet (GFD), and 11 controls), were recruited at the Paediatrics Department (University of Naples Federico II). The expression of 365 human miRNAs was quantified by TaqMan low-density arrays. We used bioinformatics to predict putative target genes of miRNAs and to select biological pathways. The presence of NOTCH1, HES1, KLF4, MUC-2, Ki67 and beta-catenin proteins in the small intestine of CD and control children was tested by immunohistochemistry. The expression of about 20% of the miRNAs tested differed between CD and control children. We found that high miR-449a levels targeted and reduced both NOTCH1 and KLF4 in HEK-293 cells. NOTCH1, KLF4 signals and the number of goblet cells were lower in small intestine of children with active CD and in those on a GFD than in controls, whereas more nuclear beta-catenin staining, as a sign of the WNT pathway activation, and more Ki67 staining, as sign of proliferation, were present in crypts from CD patients than in controls. In conclusion we first demonstrate a miRNA mediated gene regulation in small intestine of CD patients. We also highlighted a reduced NOTCH1 pathway in our patients, irrespective of whether the disease was active or not. We suggest that NOTCH pathway could be constitutively altered in the celiac small intestine and could drive the increased proliferation and the decreased differentiation of intestinal cells towards the secretory goblet cell lineage.	[ { "section_content": "Celiac disease (CD) is an immunomediated enteropathy and one of the most heritable complex diseases.The concordance rate in monozygotic twins is 75% [1,2].HLA DQ2/DQ8 haplotypes confer the highest estimated heritability (,35%) [3] reported so far. Exposure to gliadin triggers an inappropriate immune response in HLA-susceptible individuals.However, the presence of HLArisk alleles is a necessary but not sufficient condition for the development of CD.In fact, about 30-40% of healthy subjects carry HLA-risk alleles [4,5].Attempts at identifying non-HLA major genetic risk loci have been unsuccessful [6].Gluten has also been shown to affect epithelial differentiation-associated genes in the small intestinal mucosa of celiac patients [7,8].However, the role of miRNA-based regulatory mechanisms in mediating gene expression alteration in CD has not yet been investigated.MicroRNAs (miRNAs) are small non-coding RNAs, 20-25 nt long, that modulate gene expression through canonical base pairing to complementary sequences in the 39UTR of target mRNA [9].Since their identification in 1993 [10], miRNAs have been found to play a relevant role in regulating gene expression in a variety of biological processes in physiological and pathological conditions [11] including autoimmune disorders [12].They can be involved in the development of mature immune cells and in the control of their function [13][14][15].MiRNAs are also important in the differentiation and function of the mouse intestinal epithelium [16]. In this study, we evaluated the miRNA expression pattern in the small intestine of children affected by active CD, children with CD on a gluten-free diet (GFD) and control children without CD.Our aim was to look for miRNA-based modulation of gene expression in celiac small intestine, and particularly for genes involved in cell intestinal differentiation/proliferation mechanisms. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Clinical features of our CD patients and controls are reported in Table 1.Villous atrophy was subtotal or total [TIIIB: n = 3 (15%) and TIIIC: n = 17 (85%)] in all active CD patients.Only minor histological abnormalities were present in GFD patients [T0: n = 5 (56%) and TI: n = 4 (44%)] and in control patients [T0: n = 7 (64%) and TI: n = 4 (36%)]. ", "section_name": "Clinical features of CD patients and controls", "section_num": null }, { "section_content": "Figure 1 shows the miRNA expression in the small intestine of children with active CD (panel A) and in children on a GFD (panel B).Ninety of the 365 (25%) miRNAs tested were not expressed in small intestine.Over 50% of miRNAs were expressed at similar levels in the two groups of CD compared to controls.On the contrary, the expression levels of about 20% of miRNAs (22% in active CD and 19% in GFD) differed between CD and controls.In particular, in active CD patients 27 and 55 miRNAs were expressed respectively more (RQ$2.0)or less (RQ#0.5)than in controls, whereas in GFD patients 22 and 49 miRNAs were expressed respectively more (RQ$2.0)or less (RQ#0.5)than in controls.The miRNAs that were differently expressed in the two CD groups are listed in Table S1. Two sets of miRNAs (one down-regulated and one upregulated compared to controls) show similar expression levels in active and GFD CD patients, being miR-449a the highest expressed miRNA Among the miRNAs differently expressed between CD children and controls, but with similar expression levels in active and in GFD CD, 9 were up-regulated and 21 were down-regulated (Table 2).Particularly, among the down-regulated miRNAs the set of miR-124a, miR-189, miR-299-5p and miR-379, was previously reported associated with autoimmune disorders [17].Among the up-regulated miRNAs the miR-449a was expressed at very high levels in all active CD (55.18616.45mean RQ6SEM) and GFD children (15.4367.69mean RQ6SEM).qRT-PCR confirmed the expression levels both of miR-449a (active CD: 2.860.9 mean RQ6SEM) and of 2 other tested miRNAs, the down-regulated miR-124a (active CD:0.660.1 mean RQ6SEM) and the similar to control expressed miR-564 (active CD:1.460.3 mean RQ6SEM vs 1.260.1 at array). ", "section_name": "CD children and controls have a different miRNA expression levels in small intestine", "section_num": null }, { "section_content": "Six of the 11 programs [Target Scan 5.1, PicTar, Miranda 1.9, MirTarget2 (v2.0), PITA (Catalog version 3) and RNAhybrid (v2.2)], which we used to predict putative target genes of miR-449a, identified several proteins that are present in relevant biological pathways.The biological pathways predicted to be deregulated by miR-449a and sorted in functional groups are reported in Figure S1 (http://mirecords.biolead.org/interactions.php?species=Homo+sapiens &mirna_acc=hsa-miR449a&targetgene_type=refseq_acc&targetgene_ info=&v=yes&search_int=Search (http://www.targetscan.org/cgi-bin/targetscan/vert_50/targetscan.cgi?species=Human&gid=&mir_sc= &mir_c=&mir_nc=&mirg=hsa-miR-449a).Among putative target genes the programs identified NOTCH1, Krueppel-like factor 4 (KLF4), delta-like 1 (DLL1), lymphoid enhancer-binding factor 1 (LEF1) and numb homolog-like (NUMBL) proteins, which are all involved in the Notch pathway.As this pathway plays a relevant role in the control of intestinal cell fate in animal models we further examined the interaction of miR-449a with Notch pathway [18]. MiR-449a binds to the 39 UTR of NOTCH1 and KLF4 and inhibits their expression.We verified the interaction between miR-449a and the 39 UTR of NOTCH1 and of KLF4 using the luciferase reporter assay.In cells co-transfected with pRL-NOTCH1 vector and pre-miR-449a or with pRL-KLF4 vector, a pre-miR-449a concentration of 100 nmol/L was sufficient to significantly reduce (respectively, p = 0.001 and p = 0.002) Renilla luciferase activity versus control values after 48 h (Figure S2A andS2B).This finding confirms the interaction between miR-449a and the 39 UTR of both NOTCH1 and KLF4. The direct interaction between miR-449a and the 39UTRs of both NOTCH1 and KLF4 was further confirmed after mutating the putative target sites in 39UTR of the two genes (See Materials and Methods S1). NOTCH1 and HES1 mRNAs are expressed in the small intestine of CD patients NOTCH1 and HES1 mRNA levels, tested by qRT-PCR, were expressed in the small intestine of active CD patients (RQ6SEM: 3.461.3and 1.460.2,respectively vs controls) and of GFD patients (RQ6SEM: 6.564.7 and 0.760.1,respectively vs controls). ", "section_name": "Bioinformatic prediction of the target genes of miR-449a", "section_num": null }, { "section_content": "We next investigated the protein expression of NOTCH1 and of HES1, which is a well known target gene of the Notch receptor family, in small intestinal biopsies from CD patients and controls.Figure 2 shows the results obtained for NOTCH1.NOTCH1 was homogeneously distributed in the intestinal villi and crypts of controls and higher expressed in crypts of controls than in crypts of active and GFD CD patients (panel A, B). In Figure 2 (panel C) are also the images converted for automated analysis (white: unstained cells, yellow/orange: low/ moderately stained cells, brown: intensely stained cells).Significantly more intensely stained and less unstained cells (p = 0.02) were detected in controls than in the two groups of CD patients, whereas no statistical significant difference was observed between the two CD groups (Figure S3, panel A and Figure S4).These results indicate that NOTCH1 is less expressed in the small intestine of active and GFD CD patients than in controls. Figure 3 shows the results obtained for HES1.HES1 was homogeneously distributed in the intestinal villi and crypts of controls and higher expressed in crypts of controls than in crypts of active and GFD CD patients (panels A and B).In Figure 3 (panel C) are also the images converted for automated analysis (white: unstained cells, yellow/orange: low/moderately stained cells, brown: intensely stained cells).Significantly more intensely stained cells were detected in controls than in CD patients (p = 0.02) and significantly less unstained cells were detected in controls than in active CD patients (p = 0.03), whereas no statistical significant difference was observed between the two CD groups (Figure S3, panel B and Figure S5).These results indicate that HES1 is less expressed in the small intestine of active and GFD CD patients with respect to controls.The above findings confirm that NOTCH1 signaling is reduced in patients affected by CD. ", "section_name": "NOTCH1 and HES1 proteins are underexpressed in the small intestine of CD patients", "section_num": null }, { "section_content": "We also investigated the protein expression of KLF4, another selected target gene of miR449a, in small intestinal villi from GFD patients and controls, lacking the villous architecture in active CD patients.We found that the levels of this protein (mean6SEM) were significantly lower in villi from GFD patients vs controls, respectively 29.065.0vs 79.063.0 (p,0.0001)(Figure S6, panel A).Since KLF4 negatively regulates cellular proliferation, we examined the effect of inhibition of KLF4 on the proliferation of intestinal crypts with the proliferation marker Ki67.The results show that the number of Ki67 positive cells is higher in the crypts of CD patients than in controls (Figure S6, panel B).Because KLF4 is also involved in the differentiation and maturation of secretory goblet cells we examined these cells by immunohistochemistry and using anti-MUC-2 antibodies.We detected statistically fewer MUC-2-stained cells (mean6SEM) in the crypts of active CD patients (18.061.6) and GFD patients (15.063.0)than in controls (35.067.7)(p = 0.04) (Figure 4A andB).Moreover, there were fewer goblet cells in the villi of GFD B).Data are expressed as percentage of miRNAs tested (n = 365).White areas, miRNAs whose expression levels were similar in the two CD groups and controls; gray areas, miRNAs not expressed; black areas, miRNAs whose expression levels differed between CD patients and controls (up-regulated q(RQ$2.0)or down-regulated Q(RQ#0.5)).doi:10.1371/journal.pone.0029094.g001patients (7.061.8)than in controls (20.064.9)(p = 0.04) (data not shown).This finding demonstrates that the differentiation of the secretory goblet cells is impaired in small intestine of CD patients. ", "section_name": "KLF4 protein is reduced and the number of goblet cells is significantly lower in the small intestine of CD patients versus controls", "section_num": null }, { "section_content": "Because NOTCH1 and also KLF4 interact with the WNT pathway to influence the intestinal stem cell fate, we investigated the WNT pathway using beta-catenin antibodies.By counting the beta-catenin positive nuclei/crypt for each patient we observed higher even if not statistical significant mean percentage betacatenin positive nuclei/crypt in active CD and GFD patients than in controls, respectively 57.0611.5 and 37.064.6 vs 27.064.6(Figure S7).This finding suggests that cellular proliferation is increased in the small intestine of CD patients. ", "section_name": "Expression of beta-catenin", "section_num": null }, { "section_content": "A very recent study established the importance of miRNAs in the differentiation and function of the mouse intestinal epithelium [16], whereas there are no data about miRNAs expression in human CD small intestine.Our study reveals that the expression of about 20% of miRNAs tested in the small intestine differed among CD and control children irrespective of whether the disease was active or not.Particularly, the miR-449a showed the highest expression level in CD patients than in controls.The miR-449 (a and b) cluster is embedded into an intronic sequence of the mRNA-encoding gene CDC20B on Chr 5q11.2 [17].MiR-449a seems to be regulated through activation of its host gene, CDC20B, and both were induced by the cell cycle regulator E2F1 [19].The mature miR-449a sequence is evolutionarily conserved across a variety of species (monkey, horse, rodents, and dogs) and therefore it probably exerts an important function [20].The bioinformatics search for putative target genes of miR-449a revealed about one hundred proteins, among these several belonged to the Notch pathway, i.e., NOTCH1, KLF4 (a NOTCH1 transcription factor) [21], DLL1, LEF1 and NUMBL.Our strategy to choose NOTCH1 gene among the other putative miRNA-target genes was based on many studies highlighting that cellular formation of the villi in small and large intestine is affected by signaling pathways such as Notch, Wnt and BMP [22][23][24][25].Notably, deregulation of the intestinal epithelial formation has been reported in several intestinal diseases such as Crohn, ulcerative colitis and colon cancer [26].Further, NOTCH1 and KLF4 genes are both involved in the control of mouse intestinal epithelial homeostasis [18,27].In fact, in mouse intestine, also in cooperation with WNT signals, NOTCH1 guides cell proliferation and differentiation [18] and KLF4 inhibition by NOTCH1 or KLF4 deletion was shown to reduce the differentiation and maturation of goblet cells [21,[27][28][29].The Notch family is constituted by single transmembrane receptors that, in mammals, after interaction with ligands (DLL1,3,4 and Jagged 1-2) undergo proteolytic cleavage and finally translocate into the nucleus where they activate target gene transcription [30]. After confirmed the interaction between miR-449a and both NOTCH1 and KLF4 mRNA, we measured the NOTCH1 and KLF4 protein levels in small intestinal biopsies of CD children.NOTCH1-positive cells were significantly fewer in biopsies from active and GFD CD patients versus controls.Similar results were also obtained for HES1, a target gene of NOTCH1 [31].Globally, these data indicate that the NOTCH1 pathway is deregulated in intestinal epithelium of CD children, irrespective of whether the disease is active or not, and that this alteration could be related to the very high miR-449a expression.Accordingly, in a very recent report miR449 by repressing the Delta/Notch pathway was elegantly shown to control the human airway epithelium and vertebrate multilciliogenesis [32].In our patients we also observed fewer KLF4-positive cells in small intestinal villi from GFD patients than in controls, and, moreover, Ki67 signals were higher in crypts from CD patients versus controls.These two results are in agreement with data very recently reported in a mouse KLF4 DELTAIS model [27] and indicate a higher proliferation rate in our CD patients in the presence of reduced KLF4 expression [27].In parallel the number of goblet cells was significantly lower in the two CD groups than in control children.Ciacci et al [33] previously reported fewer goblet cells/mm 2 in untreated (29.1) and in treated CD patients (42.2) than in controls (50.5),although the differences were statistically significant only in untreated patients (p,0.02). The WNT pathway in the small intestine of our CD patients, evaluated based on beta-catenin expression level, did not differ from that of control children.This result is in agreement with the western blot data reported by Ciccocioppo et al [34] and by Juuti-Uusitalo et al [7].However, we observed a more evident nuclear localization of beta-catenin, albeit not statistically significant, in the small intestinal crypts from our active and GFD CD patients than in controls, which suggests activation of the WNT pathway.The latter finding is in agreement with a previous study of human CD [7] and with the increased mRNA levels of the genes in the WNT pathway, including beta-catenin, observed in the KLF4 DELTAIS mouse [27].Globally, our data support increased cellular proliferation in the small intestinal epithelium in CD patients.As it is well known, active CD is characterized by an inversion of the differentiation/proliferation program of the intestine with a reduction in the differentiated compartment, up to complete villous atrophy, and an increase of the proliferative compartment, with crypt hyperplasia [7,8].Furthermore, although GFD intestinal mucosa is characterized by an apparently normal mucosal architecture, it can also be associated with increased crypt cell proliferation (Barone M. V. et al., personal communication).Our data are in contrast with those obtained in mouse models, in which NOTCH1 activation resulted in a reduction of goblet cells consequent to HES-1 dependent repression of Math1 (intestinal secretory cell differentiation factor) [18] and in which NOTCH1 inhibited the expression of KLF4 [35].However, our data are in agreement with a recent report of increased proliferation, reduced differentiation and goblet cells maturation associated with downregulation of the expression of components of the Notch pathway (HES1, DLL1, JAG1) in the small intestine of the KLF4 DELTAIS mouse [27].The authors for the latter article hypothesized that KLF4 was involved in a feed-back loop by positively regulating Notch signaling.Our results are suggestive that an altered NOTCH1 and KLF4 expression could lead to the reduction of goblet cells in the small intestine of CD patients.The maintenance of a correct number of functional goblet cells is required for the homeostasis of the intestinal mucosal environment, and deficiencies in the mucin composition renders the mucosa more susceptible to damaging agents in the lumen [36][37][38].In fact, loss of goblet cell function leads to spontaneous colitis in mice [39].Moreover, an altered mucous layer and increased rod-shaped bacteria and interferon-gamma mRNA levels were found in intestine from CD patients [40].Based on these experimental data, we suggest that the mucus layer in our CD children could be altered so deranging the protective function of the mucosal barrier that interfaces with the environment.In our study, the observed small intestine alterations are not related to inflammation; in fact, they occurred in both the active CD and GFD patients.The major criticism in our work is the gap between the results of the miRNA array with NOTCH1 gene in a vivo system, however the lack of a celiac animal model at moment, prevent us from this further validation of our data.Nevertheless, our first description of miRNA pattern in celiac disease and of the correlation of miRNA 449a over expression with NOTCH pathway could pave the way for further research in this field.However, our choice to study Notch pathway doesn't exclude that other relevant biological pathways in addition to it could be miRNA-deregulated in the celiac intestine.Further deeper investigation are necessary to test this hypothesis. In conclusion we first demonstrate a miRNA mediated gene regulation in small intestine of CD patients.We also highlighted a reduced NOTCH1 pathway in our patients, irrespective of whether the disease was active or not.We suggest that NOTCH pathway could be constitutively altered in the celiac small intestine and could drive the increased proliferation and the decreased differentiation of intestinal cells towards the secretory goblet cell lineage. ", "section_name": "Discussion", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "The study was conducted according to the Helsinki II declaration and it was approved by the Ethics Committee of the School of Medicine Federico II, Naples, Italy. Written informed consent was obtained from the parent/guardian of all children involved in our study before their enrollment.follow-up in the same study period of the active CD patients and controls.There was no statistically significant difference in mean age at diagnosis among the groups evaluated (4.361.3 years old in active CD subjects, 7.662.5 in GFD subjects, and 6.161.0 in controls [mean6SEM]).About 50% of each group was girls.From all participants, we collected a fasting serum sample, a blood sample with EDTA, and a small intestine biopsy sample. ", "section_name": "Ethics approval", "section_num": null }, { "section_content": "", "section_name": "Patients and controls", "section_num": null }, { "section_content": "Anti-Endomysium IgA were detected by indirect immunofluorescence on rhesus monkey esophagus substrate (Eurospital, Trieste, Italy); tTG IgA, anti-gliadin (AGA) IgA/IgG were analyzed by ELISA with human recombinant tTG as antigen (DIA Medix Corp., Miami, FL, USA). ", "section_name": "Biochemical parameters", "section_num": null }, { "section_content": "Architectural abnormalities were classified according to the modified Marsh classification: normal mucosa (T0), intraepithelial lymphocytosis (TI), intraepithelial lymphocytosis and crypt hyperplasia (TII), intraepithelial lymphocytosis, crypt hyperplasia and villous atrophy (partial TIIIA, subtotal TIIIB, total TIIIC) [42]. ", "section_name": "Histopathological analysis", "section_num": null }, { "section_content": "Genomic DNA was extracted using the Nucleon BACC 2 kit (Amersham Biosciences Europe, Milan, Italy).Total RNA, including miRNAs, was extracted from small intestinal biopsy samples using the Mirvana extraction kit (Applied Biosystems, Foster City, CA, USA). ", "section_name": "DNA and RNA extraction", "section_num": null }, { "section_content": "DQ2/DQ8 HLA CD-associated molecules were identified by using primers and the PCR conditions of a commercial kit (BAG Health care GmbH, Lich, Germany), which allows to identify the HLA-alleles coding DQ2/DQ8 molecules. ", "section_name": "HLA typing", "section_num": null }, { "section_content": "TaqMan low density arrays (TLDA), micro fluidic cards were used to detect and quantify mature miRNAs (Applied Biosystems' 7900HT) according to manufacturer's instructions (see Materials and Methods S1 for details).We considered differently expressed in CD vs controls, the miRNAs whose mean RQ levels were #0.5 (down-regulated) or $2.0 (up-regulated). ", "section_name": "MiRNAs evaluation", "section_num": null }, { "section_content": "The prediction of putative target genes of miRNAs was determined using miRecords (http://mirecords.biolead.org/),which is an integration of 11 established miRNA target prediction programs.The lists of target genes that were predicted by two or more programs were then combined and analyzed using the Gene Ontology Tree Machine (GOTM) (http://bioinfo.vanderbilt.edu/gotm/) and KEGG database (http://www.genome.ad.jp/kegg/).Finally, we identified the biological pathways that contained at least two up-or down-regulated genes with a statistically significant probability (p,0.01). ", "section_name": "Bioinformatic approach", "section_num": null }, { "section_content": "The levels of a group of deregulated miRNAs (up-regulated miR-449a, down-regulated miR-124a, and similar to controls expressed miR-564) were also evaluated with TaqMan miRNA assays (Applied Biosystems) to validate the array results. mRNA expression levels of neurogenic locus notch homolog protein 1 (NOTCH1) and of hairy and enhancer of split 1 (HES1) were measured in small intestinal tissues by qRT-PCR using single TaqMan mRNA assays (Applied Biosystems) according to the manufacturer's instructions and using the housekeeping gene betaactin as control.Reverse transcription reactions were performed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).The expression levels of miRNAs and mRNAs were quantified using the ABI Prism 7900HT Sequence Detection System 2.3 software. ", "section_name": "Quantitative real-time polymerase chain reaction (qRT-PCR) of miRNAs and mRNAs", "section_num": null }, { "section_content": "The oligonucleotides, plasmids (pGL3-control, pRL-NOTCH1encoding, pRL-KLF4-encoding and mutated pR-KLF4-encoding, firefly luciferase and Renilla luciferase, respectively) and human embryonic kidney cell lines (HEK293 cell line, ATCC number CRL-1573, supplied by the Centre for Applied Microbiology and Research, Salisbury, Wiltshire, UK) used for cell transfection experiments are described in detail in the online Materials and Methods S1. Forty-eight hours after transfection, we measured firefly and Renilla luciferase activities using a dual luciferase assay according to the manufacturer's instructions (Promega, Naples, Italy). ", "section_name": "Transfection and inhibition experiments", "section_num": null }, { "section_content": "Given the small amount of sample available for each patient (1-2 mg of intestinal tissue/patient) we tested the expression of selected proteins by immunohistochemistry instead of by western blotting.The NOTCH1, HES1, MUC-2, KLF4, Ki67 and betacatenin proteins were identified on formalin-fixed paraffinembedded small intestinal tissue blocks in CD patients and in controls.We randomly selected six active CD, six GFD and four controls (see Materials and Methods S1 for details).We also tested the specificity of our NOTCH1 and HES1 signals evaluating two different human tissue samples where it is known NOTCH1 and HES1 be present or absent respectively, that are colon cancer and endothelial wall (Figure S8). ", "section_name": "Protein evaluation by immunohistochemistry", "section_num": null }, { "section_content": "To increase precision, we automated the quantification of the immunohistochemical signals.Sections of the small intestine were scanned with the NanoZoomer 2.0 system (Hamamatsu, Japan), equipped with a 206, 0.7 Numerical Aperture Plan-Apochromat lens, using a lens of 0.23 mm pixel size.The compressed jpeg files were transferred to the Definiens Analyst LS5.0 system (Definiens AG, Germany) that counted the NOTCH1, HES1 and betacatenin -positive and -negative cells and quantified the staining signal (see materials and methods S1 for Definiens Analyst software details).The Definiens Analyst software (Definiens AG, Germany) is based on cognition network technology that is a semantic network of objects and their mutual relationships.Two rule sets, using cognition network language, were specifically written for this evaluation to automatically detect and measure the small intestinal area and to count positive and negative crypt cells.The signal was classified as intensely stained, low/moderate stained and unstained.Thus, both the percentage and intensity of labeled cells were taken into account.The detection and exclusion of areas not belonging to crypt were visually checked for all image files.Ten crypts/patient were counted. ", "section_name": "Scanning and automated image analysis of NOTCH1 and HES1", "section_num": null }, { "section_content": "Because the MUC-2 staining of goblet cells was patchy, we picked ten crypts from each slide and manually counted the number of goblet cells stained in each crypt.We also evaluated MUC-2 staining of villi, when possible, i.e., in GFD patients and controls.We also evaluated KLF4-positive villi (in GFD patients) and both beta-catenin-and Ki67-positive nuclei/crypt in each subject.Two independent observers evaluated the immunohistochemical slides. ", "section_name": "Immunohistochemical analysis of MUC-2, KLF4 and betacatenin", "section_num": null }, { "section_content": "All variables were expressed as mean6standard error of the mean (SEM).Student t's test and ANOVA were used to compare group means and p values,0.05 were considered significant.Statistically significant (p,0.01)miRNA-regulated pathways were selected by the GOTM program. ", "section_name": "Statistics", "section_num": null } ]	[ { "section_content": "Jean Ann Gilder (Scientific Communication srl) provided writing assistance. Provenance and peer review not commissioned; externally peer reviewed. This paper is dedicated to Prof. Salvatore Auricchio.He has promoted research in celiac disease in Naples for 40 years and kindly read and commented on this study.We are grateful to M. V. Barone for helpful discussion and to Carolina Tarantino for skilled technical assistance. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "Study supported from European community (PREVENT CD project: EU-FP6-2005-FOOD4B-contract no.036383).The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "Materials and Methods S1 (DOC) Figure S1 Bioinformatics analysis of miR-449a putative target genes.miR-449a putative target genes with most favorable context score, selected by bioinformatics, were sorted into pathways using GOTM(http://bioinfo.vanderbilt.edu/webgestalt/)and then combined into functional groups.(http://mirecords.biolead.org/interactions.php?species=Homo+sapiens&mirna_acc =hsa-miR-449a&targetgene_type=refseq_acc&targetgene_info=& v=yes&search_int=Search) (http://www.targetscan.org/cgibin/targetscan/vert_50/targetscan.cgi?species=Human&gid=&mir_sc =&mir_c=&mir_nc=&mirg=hsa-miR-449a).In each functional group are reported the genes belonging to NOTCH pathway/ total gene number.(TIF) The luciferase assay confirms that miR-449a inhibits the expression of NOTCH1 and KLF4.In HEK293 cells co-transfected or with pRL-NOTCH1 vector (panel A) or with pRL-KLF4 vector (panel B), a pre-miR-449a concentration of 100 nmol/L was sufficient to significantly reduce (respectively, p = 0.001 and p = 0.002) Renilla luciferase activity versus control values.No inhibition of the Renilla luciferase expression was observed in mutant 39UTR of KLF4-mRNA with miR-449a, so confirming the miR-449a/39UTR KLF4-mRNA direct interaction (panel B).We didn't verify the interaction miR-449a/39UTR NOTCH1 being this latter recently validated by Marcet B et al [32].(TIF) Figure S3 Automated Counts of NOTCH1 and HES1 stained/unstained cells.A. Automated counts of NOTCH1 stained/unstained cells (reported in Figure 2) in small intestine from CD patients (6 active CD and 6 GFD patients) and from controls (n = 4).Data are expressed as mean percent of intensely stained, low-moderately stained and unstained cells of the total intraepithelial cells (IECs) counted in ten crypts.The numbers of intensely stained and unstained cells were significantly (p = 0.02) higher and lower, respectively, in CTRL than in active CD and in GFD patients.B. Automated counts of HES1 stained/ unstained cells (reported in Figure 3) in small intestine from CD patients (6 active CD and 6 GFD patients) and from controls (n = 4).Data are expressed as mean percent of intensely stained, low-moderately stained and unstained cells of the total intraepithelial cells (IECs) counted in ten crypts.The number of intensely stained cells was significantly higher in controls versus CD and GFD patients (p = 0.02) and the number of unstained cells was significantly lower in CTRL than in active CD patients Immunostaining with beta-catenin in small intestinal crypts from active CD, GFD and controls.We counted the beta-catenin labeled nuclei.Similar counts of beta-catenin labelled nuclei were detected in the crypts of the small intestine in all groups.However, higher even if not statistical significant mean percentage counts (beta-catenin positive nuclei/crypt) were obtained in active CD and GFD than in controls, respectively 57.0611.5 and 37.064.6 vs 27.064.6(Original magnification 636).(CTRL: controls; GFD: gluten free diet; CD: celiac disease).(TIF) ", "section_name": "Supporting Information", "section_num": null }, { "section_content": "Materials and Methods S1 (DOC) Figure S1 Bioinformatics analysis of miR-449a putative target genes.miR-449a putative target genes with most favorable context score, selected by bioinformatics, were sorted into pathways using GOTM(http://bioinfo.vanderbilt.edu/webgestalt/)and then combined into functional groups.(http://mirecords.biolead.org/interactions.php?species=Homo+sapiens&mirna_acc =hsa-miR-449a&targetgene_type=refseq_acc&targetgene_info=& v=yes&search_int=Search) (http://www.targetscan.org/cgibin/targetscan/vert_50/targetscan.cgi?species=Human&gid=&mir_sc =&mir_c=&mir_nc=&mirg=hsa-miR-449a).In each functional group are reported the genes belonging to NOTCH pathway/ total gene number.(TIF) ", "section_name": "Supporting Information", "section_num": null }, { "section_content": "The luciferase assay confirms that miR-449a inhibits the expression of NOTCH1 and KLF4.In HEK293 cells co-transfected or with pRL-NOTCH1 vector (panel A) or with pRL-KLF4 vector (panel B), a pre-miR-449a concentration of 100 nmol/L was sufficient to significantly reduce (respectively, p = 0.001 and p = 0.002) Renilla luciferase activity versus control values.No inhibition of the Renilla luciferase expression was observed in mutant 39UTR of KLF4-mRNA with miR-449a, so confirming the miR-449a/39UTR KLF4-mRNA direct interaction (panel B).We didn't verify the interaction miR-449a/39UTR NOTCH1 being this latter recently validated by Marcet B et al [32].(TIF) Figure S3 Automated Counts of NOTCH1 and HES1 stained/unstained cells.A. Automated counts of NOTCH1 stained/unstained cells (reported in Figure 2) in small intestine from CD patients (6 active CD and 6 GFD patients) and from controls (n = 4).Data are expressed as mean percent of intensely stained, low-moderately stained and unstained cells of the total intraepithelial cells (IECs) counted in ten crypts.The numbers of intensely stained and unstained cells were significantly (p = 0.02) higher and lower, respectively, in CTRL than in active CD and in GFD patients.B. Automated counts of HES1 stained/ unstained cells (reported in Figure 3) in small intestine from CD patients (6 active CD and 6 GFD patients) and from controls (n = 4).Data are expressed as mean percent of intensely stained, low-moderately stained and unstained cells of the total intraepithelial cells (IECs) counted in ten crypts.The number of intensely stained cells was significantly higher in controls versus CD and GFD patients (p = 0.02) and the number of unstained cells was significantly lower in CTRL than in active CD patients Immunostaining with beta-catenin in small intestinal crypts from active CD, GFD and controls.We counted the beta-catenin labeled nuclei.Similar counts of beta-catenin labelled nuclei were detected in the crypts of the small intestine in all groups.However, higher even if not statistical significant mean percentage counts (beta-catenin positive nuclei/crypt) were obtained in active CD and GFD than in controls, respectively 57.0611.5 and 37.064.6 vs 27.064.6(Original magnification 636).(CTRL: controls; GFD: gluten free diet; CD: celiac disease).(TIF) ", "section_name": "Figure S2", "section_num": null } ]
10.18632/oncotarget.8381	Lysine demethylase 2A promotes stemness and angiogenesis of breast cancer by upregulating Jagged1	Alterations of histone methylation dynamically regulated by methyltransferases and demethylases are frequently found in human cancers. Here, we showed that expression of lysine demethylase 2A (KDM2A) is markedly increased in human breast cancer and its overexpression is associated with tumor progression and poor prognosis. Knockdown of KDM2A in breast cancer cells reduced proliferation but not viability. Gene set enrichment analysis revealed that inhibition of KDM2A down-regulates angiogenic genes with concurrent reduction of Jagged1 (JAG1), NOTCH1 and HEY1 in the NOTCH signaling. Chromatin immunoprecipitation- quantitative polymerase chain reaction (ChIP-qPCR) demonstrated the binding of KDM2A to the JAG1 promoter and the increase of methylation of Lys-36 of histone H3 (H3K36) in KDM2A-depleted MDA-MB-231 cells. Tumorsphere formation was significantly reduced in KDM2A-depleted cells which could be reversed by ectopic expression of JAG1. A selective KDM2A inhibitor daminozide also decreased the number of tumorsphere and the number of CD24-/CD44hi cells. In addition, daminozide acted synergistically with cisplatin in cell killing. We identified SOX2 as a direct transcriptional target of KDM2A to promote cancer stemness. Depletion of KDM2A in MDA-MB-231 cells attenuated NOTCH activation and tube formation in co-cultured endothelial cells. Two pro-angiogenic factors JAG1 and PDGFA are key mediators for KDM2A to enhance angiogenesis. Finally, inhibition of KDM2A significantly decreased tumor growth and angiogenesis in orthotopic animal experiments. Collectively, we conclude that KDM2A functions as an oncogene in breast cancer by upregulating JAG1 to promote stemness, chemoresistance and angiogenesis.	[ { "section_content": "Histone proteins are the building components of the nucleosome, the basic unit of DNA packaging in eukaryotic cells.Histone proteins undergo different post-translational modifications (PTMs) including phosphorylation, acetylation, methylation, sumoylation and ubiquitination.These modifications affect the compact of chromatin structure and the interactions between non-histone proteins and chromatin to modulate gene expression.The ultimate effects of histone modification on gene transcription dependent on the type of modification, the modified residues, and the degree of modification [1][2][3].Tri-methylation of lysine 4 of histone H3 (H3K4) is strongly associated with transcriptional activation and is frequently found around the transacription start sites of highly expressed genes, whereas H3K27 tri-methylation is usually linked with transcriptional inhibition and is detected in the promoters of silenced genes [4][5][6].In contrast to the repressive role of H3K27 tri-methylation, recent study suggested that mono-methylated H3K27 accumulates within active genes and promotes transcriptional activation [7]. Methylation of histone proteins is tightly regulated by histone methyltransferases and demethylases [8].Lysine demethylase 2A (KDM2A) was originally cloned as a member of mammalian F-box protein families that are critical components of the SCF ubiquitin-protein ligase complexes [9].Subsequently, another group cloned a novel gene CXXC8 (also known as FBXL11) as a new member of CXXC family genes [10].Tsukada et al purified a JmjC domain-containing protein JHDM1 which acts as a H3K36 demethylase by using Fe2+ and α-ketoglutarate as cofactors [11].It turns out that these three molecules are encoded by the same gene, and is now officially named as KDM2A.Among the methylation sites of histone H3 studied, the biological significance of H3K36 is relatively unclear.H3K36 methylation has been associated with transcription activation.However, the genetic location where the methylated H3K36 is added, the timing when H3K36 is methylated, and the degree of methylation all determine the final biological outcomes [12]. The potential role of KDM2A in carcinogenesis has been demonstrated recently in lung cancer [13].KDM2A was identified as one of the most upregulated histone demethylases in lung cancer.This demethylase induced H3K36 demethylation of the promoter region of the dual-specificity phosphatase 3 (DUSP3) gene that led to down-regulation of DUSP3.Because DUSP3 is an important dephsophorylating enzyme of extracellular signal-regulated kinases (ERKs), reduced expression of DUSP3 increased ERK activation and promoted cell proliferation and invasiveness.Results of the study suggested KDM2A is an oncogene in lung cancer.However, the contribution of KDM2A to other cancers is largely unknown.In addition, the downstream mediators by which KDM2A promotes carcinogenesis is also unclear.In the present study, we investigated the expression of KDM2A in breast cancer tissues and examined its association with clinicopathological features. In addition, we tried to elucidate how KDM2A regulates the behaviors of breast cancer cells and identified JAG1 as a key downstream effector for KDM2A to promote stemness and angiogenesis in breast cancer. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "We performed immunohistochemical staining to investigate the expression of KDM2A in 202 breast tumor tissues.As shown in Figure 1A, fifty percent (101/202) of tumor tissues exhibited high expression of KDM2A with strong nuclear staining and light cytoplasmic staining.Clinicopathological association study demonstrated that high KDM2A is significantly correlated with large primary tumor, increased lymph node metastasis, advanced stage and high histological grade (Table 1).Kaplan-Meier survival analysis demonstrated that patients with high KDM2A expression have a short disease-specific survival (Figure 1B) and metastasis-free survival (Figure 1C).Univariate log-rank analysis showed tumor size, nodal status, stage and KDM2A expression are associated with the survival of breast cancer patients (Table 2).In addition, high KDM2A expression appeared as an independent prognostic factor for disease-specific survival (hazard ratio=5.406,95% CI=1.447 to 20.201, P=0.012) and metastasis-free survival (hazard ratio=3.476,95% CI=1.727 to 6.998, P<0.001) in multivariate analysis (Table 3).These data suggested that breast cancer patients with KDM2A up-regulation have poor prognosis.To validate our conclusion, we did bioinformatics analysis by using the public databases of PM Plotter [14] and PROGgeneV2 [15].By using the Jetset best probe #208988 and auto select cutoff value for analysis, high KDM2A expression is associated poor survival (hazard ratio=1.15(1.02-1.29)and p=0.019) (Figure 1D).Similarly, the PROGgeneV2 database indicated a poor overall survival of breast cancer patients with high KDM2A expression (p=0.0425)(Figure 1E). ", "section_name": "Upregulation of KDM2A in breast cancer is associated with short survival", "section_num": null }, { "section_content": "We next investigated the expression of KDM2A in breast cancer cell lines.As shown in Figure 2A, KDM2A protein level is very low in normal human mammary epithelial M10 cells and a 5.1-, 8.3-, and 7.5-fold increase was detected in MCF-7, SkBr3 and MDA-MB-231 cells respectively.To investigate the effect of KDM2A on cancer cell behaviors, we generated two independent KDM2A-depleted stable clones (231-3A1 and 231-2A2) from MDA-MB-231 cells by shRNA knockdown (Figure 2B).Gene expression profiles of two KDM2Adepleted clones are distinct from that of parental cells (Figure 2C).Gene set enrichment assay (GSEA) analysis indicated that the most significantly changed pathways are related to DNA replication and cell cycle regulation (Figure 2D).Interestingly, in the top 25 core genes in the DNA replication geneset obtained by GSEA, both cell cycle promoters (like CCNA2, CDC45 and AURKB) and inhibitors (like RB1, GMNN and PPP2CB) are upregulated in KDM2A-depleted cells (Figure 2E).Therefore, the effect of KDM2A depletion in breast cancer cells was further investigated.We found that the proliferation of MDA-MB-231-2A2 cells was reduced (Figure 2F).The doubling time of MDA-MB-231 cells is 23.1 h while it increased to 36.8 h in MDA-MB-231-2A2 cells.Flow cytometry analysis revealed the increase of cells at the G0/G1 phase and a reduction of cells at the S and G2/M phases (Figure 2G).Because the decrease of S-phase cells is minor, we thought KDM2A depletion delays the G1 progression but not completely blocks cell cycle transition.To test our hypothesis, we synchronized cells at the G2/M phase by nocodazole and then released the cells for cell cycle analysis at different times.At 5 h after nacodazole release, the percentage of cells at the G1 phase is similar in MDA-MB-231 (62.6%) and MDA-MB-231-2A2 (65.5%) cells (Figure 2H).At 10 h, the percentage of the G1 phase of MDA-MB-231 cells was 56.5% while it increased to 69.2% in MDA-MB-231-2A2 cells.At 15 h, 35.2% of MDA-MB-231 cells and 46.7% of MDA-MB-231-2A2 cells were at the G1 phase (Figure 2H).I harvested the cells and detected the level of G1 and S phase cyclins.As shown in the low panel of Figure 2H, treatment of nocodazole inhibited cell cycle at the G2/M phase with a dramatic increase of the mitotic cyclin B. After releasing nocodazole inhibition for 15 h, cyclin B was reduced in MDA-MB-231 cells and two S phase cyclins (cyclin E and A) were significantly increased indicating these cells entered the S phase.Conversely, MDA-MB- ", "section_name": "Inhibition of KDM2A induces G1 progression delay and reduces proliferation in breast cancer cells", "section_num": null }, { "section_content": "In addition to alteration in cell proliferation, our GSEA analysis demonstrated that inhibition of KDM2A attenuates tumor angiogenesis and mRNA expression of several genes in the NOTCH signaling pathway including JAG1, NOTCH1, HEY1 (Figure 3A).Because JAG1 is the ligand for NOTCH1, we investigated whether KDM2A depletion reduces JAG1 expression and found that it is indeed the case (Figure 3B).Ectopic expression of KDM2A in MDA-MB-231-2A2 cells fully rescued the downregulation of JAG1 indicating KDM2A is an upstream regulator of JAG1 (Figure 3C).In addition, ChIP-qPCR assay demonstrated that KDM2A directly bound to the JAG1 promoter and the binding was significantly reduced in MDA-MB-231-2A2 cells (Figure 3D).Consequently, di-methylation and tri-methylation of hisone H3 lysine-36 (H3K36me2 and H3K36me3) in the JAG1 promoter is increased.In consistent with the reduction of JAG1 expression, the gene activation marker H3K4 was significantly decreased (Figure 3D).We found that PDGFA is also a direct transcriptional target of KDM2A.The mRNA level of PDGFA and the secreted PDGFA protein were reduced in KDM2A-depleted cells (Figure 3E).ChIP-qPCR assay demonstrated the direct binding of KDM2A to the PDGFA promoter (Figure 3F).In KDM2A-depelted cells, di-methylation of H3K36 of the PDGFA promoter was increased and the gene activation marker H3K4 was decreased (Figure 3F).Additionally, ectopic expression of KDM2A reversed PDGFA expression in KDM2A-depleted cells (Figure 3G). To rule out the cell line-specific effect, we inhibited KDM2A in SkBr3 breast cancer cells and found the expression of JAG1 and PDGFA was also reduced (Figure 4A and4B).To confirm the clinical relevance, we performed bioinformatics analysis of a public database (GSE2034) with the gene expression profiles of 286 breast cancer patients.We found a strong positive correction (P<0.0001) between KDM2A and JAG1 in these cancer patients (Figure 4C).These data suggested that JAG1 is a direct target of KDM2A to promote the activation of NOTCH1. ", "section_name": "KDM2A knockdown attenuates JAG1 expression and NOTCH1 activation", "section_num": null }, { "section_content": "The NOTCH1 signaling pathway plays a crucial role in the maintenance of stemness of normal and cancer stem cells.Breast cancer cells with stem-like properties are slow-dividing and relatively quiescent within a proliferating population.These cancer stem cells could retain the lipophilic dye PKH26 for a long time after labeling [16].We stained MDA-MB-231 cells with PKH26 and then seeded the cells onto low attachment plates for sphere formation assay.The fluorescent intensity was retained in the tumorspheres indicating (Continued ) the cancer stem-like properties of the cells (Figure 5A).Knockdown of KDM2A reduced the sphere formation of MDA-MB-231 cells and ectopic expression of JAG1 fully reversed the reduction of the tumorspheres in MDA-MB-231-2A2 cells (Figure 5B).We collected the tumorspheres after 14 days and repeated the sphere formation assay.As shown in Figure 5B, the second sphere formation was also inhibited by KDM2A depletion.Similarly, a KDM2A chemical inhibitor daminozide reduced JAG1 expression and strongly inhibited the sphere formation of MDA-MB-231 cells (Figure 5C).Breast cancer stem cells express high CD44 and are negative for CD24.We found that the population of CD24 -/CD44 hi cells was reduced in MDA-MB-231-2A2 cells and ectopic expression of JAG1 reversed the reduction (Figure 5D). Another characteristic of breast cancer stem cells is the resistance to chemotherapeutic drugs.We showed that KDM2A-depleted cells are highly sensitive to cisplatin (Figure 5E).In addition, KDM2A inhibitor daminozide significantly enhanced the cytotoxic activity of cisplatin to MDA-MB-231 cells (Figure 5F).These data suggested that inhibition of KDM2A reduces stemness and chemoresistance of breast cancer cells. ", "section_name": "KDM2A increases stemness and chemoresistance of breast cancer cells", "section_num": null }, { "section_content": "We next investigated the effect of KDM2A depletion on stemness regulators including SOX2, OCT4 and NANOG and our data showed that only SOX2 was down- regulated by KDM2A knockdown (Figure 6A).Ectopic expression of JAG1 completely rescued the reduction of SOX2 in MDA-MB-231-2A2 cells (Figure 6B).Western blot analysis demonstrated that overexpression of JAG1 does not affect KDM2A level while it reversed the expression of SOX2 suggesting JAG1 is a downstream mediator of KDM2A to upregulate SOX2 (Figure 6C).Similar results were also found in KDM2A-depleted SKBr3 cells (Figure 4A and4B).NOTCH activation induced by JAG1 leads to the generation of a NOTCH intracellular domain (NICD) which complexes with Maml and Rbpj to activate downstream transcriptional target genes via the Rbpj binding sites [17].We found high levels of NICD and SOX2 proteins in MDA-MB-231 cells (Continued ) (Figure 6D).This data is consistent with the result of a previous study showing the high level of NICD in MDA-MB-231 cells [18].Inhibition of NOTCH activation by a γ-secretase inhibitor DAPT simultaneously decreased these two proteins and also attenuated SOX2 promoter activity in MDA-MB-231 cells (Figure 6D and6E).Bioinformatics analysis revealed three Rbpj binding sites within the -1797/-875 region of human SOX2 gene promoter.Our data showed that the transcriptional activity of three SOX2 promoter constructs containing the -4057/+267, -2741/+267 and -1950/+267 promoter region are reduced in KDM-depleted cells which could be fully reversed by JAG1 overexpression (Figure 6F).Interestingly, JAG1 could not rescue the downregulation of the -1125/+267 promoter construct which lacks the Rbpj site located at the -1797/-1786 promoter region suggesting this site is important for the activation of SOX2 expression by JAG1.Mutation of this site reduced the transcriptional activity of the -1950/+267 promoter construct (Figure 6G).However, the basal activity of the -1125/+267 promoter construct is still significantly reduced in KDM2Adepleted cells.It is possible that KDM2A modulates SOX2 expression via multiple transcription regulators.Our results suggested that KDM2A upregulates JAG1 to promote NOTCH activation which directly activates the transcription of SOX2 gene in breast cancer cells. ", "section_name": "KDM2A increases the stemness regulator SOX2 via JAG1", "section_num": null }, { "section_content": "Our GSEA results showed that inhibition of KDM2A is associated with reduced angiogenesis.In addition, two core-enriched genes JAG1 and PDGFA are well-characterized pro-angiogenic factors.Therefore, we investigated the effect of KDM2A on tumor angiogenesis in vitro and in vivo.To test whether JAG1 expressed on breast cancer cells could induce tube formation in endothelial cells via direct contact, we co-cultured EA.hy926 endothelial cells with various MDA-MB-231 clones and found that co-culture of MDA-MB-231 cells but not MDA-MB-231-2A2 cells induced the formation of tube-like structure (Figure 7A).Ectopic expression of KDM2A, JAG1 or PDGFA in KDM2A-depleted MDA-MB-231-2A2 cells significantly increased their ability to induce tube formation and restored total branching and segment lengths (Figure 7B and7C). As shown in Figure 7D, knockdown of KDM2A reduced tumor weight by 60%.Abundant blood vessels were detected around the tumors generated by injection of MDA-MB-231 cells (Figure 7E).In addition, immunohistochemical staining showed strong CD31- (Continued ) positive blood vessels in the MDA-MB-231 tumors suggesting intense angiogenesis in these tumors (Figure 7F).Conversely, angiogenesis was reduced in the MDA-MB-231-2A2 tumors (Figure 7F).We also found SOX2positive cancer cells were higher in the MDA-MB-231 tumors when compared to the MDA-MB-231-2A2 tumors (Figure 7G). Figure 8 summarized the results of this study to propose a mechanistic model by which KDM2A promotes breast cancer progression. ", "section_name": "Inhibition of KDM2A attenuates tumor angiogenesis in vitro and in vivo", "section_num": null }, { "section_content": "In this study, we provide evidence that JAG1 is a direct target of KDM2A and is important for KDM2A to promote tumor stemness and angiogenesis.Previous studies demonstrated that high expression of JAG1 is frequently found in human breast cancer and is significantly associated with poor survival [19,20].In addition, aberrant activation of NOTCH signaling induced by JAG1 overexpression also predicts poor clinical outcome in breast cancer patients [20,21].However, the molecular mechanism by which JAG1 is activated in cancer cells is largely unclear.In epithelial cells from mammary gland, kidney tubule and epidermis, transforming growth factor ß could stimulate JAG1 expression via SMAD3 [22].In endothelial cells, JAG1 expression is negatively regulated by chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) to control embryonic arterial-venous differentiation [23].In early ovarian follicle development, JAG1 gene is activated by the neurotrophin-TrkB signaling pathway in oocytes [24].A recent study demonstrated that an oncogenic transcription factor Lim domain only 2 (LMO2) increases glioma stem cells by inducing JAG1 expression [25].In addition to transcriptional induction, JAG1 expression could be modulated epigenetically.By using MassARRAY spectrometry, Cao et al demonstrated that the methylation level of overall and majority individual CpG sites of the JAG1 gene promoter were significantly lower in breast tumor tissues than that of adjacent normal breast tissues [26].Because histone modification and DNA methylation are highly associated during gene transcription, it is possible that histone methylation controlled by histone methyltransferases and demethylases will affect JAG1 expression.We showed for the first time that KDM2A binds to the JAG1 promoter to increase its expression in breast cancer cells.Recently, another demethylase KDM4C has also been shown to bind onto the JAG1 promoter to mediate β-catenin-dependent transcription of JAG1 and to maintain sphere-forming capacity in colon cancer cells [27].These data suggested JAG1 is a common target of various histone demethylases to promote tumorigenesis. To clarify how KDM2A enhances stem-like properties of breast cancer cells, we investigated the alteration of stemness markers after KDM2A depletion and identified SOX2 as a key mediator.The importance of SOX2 in cancer stem cells has been highlighted recently by two independent studies.Vanner et al demonstrated that SOX2-expressing cells isolated from medulloblastoma exhibited self-renewal ability in vitro and high tumorforming activity in orthotopic animals, whereas SOX2 - cells lacked these characteristics [28].Boumahdi et al found that SOX2 + cells are absent from the normal epidermis while they are robustly increased in papilloma and squamous cell carcinoma [29].Conditional deletion of SOX2 from the epidermis significantly delayed chemical carcinogen-induced tumorigenesis.In breast cancer, expression of SOX2 was detected in early stage tumors [30].Importantly, SOX2, but not NANOG and OCT4, is upregulated in mammosphere in culture.Inhibition of SOX2 prevented the self-renewal ability and dramatically reduced mammosphere formation.Results of our study support the notion that SOX2 is a master regulator in maintaining breast cancer stem cells and provide the first evidence that SOX2 is a downstream mediator of KDM2A to promote cancer stemness. We also connect the KDM2A-increased JAG1 to SOX2 activation.Our results showed that the reduction of SOX2 in KDM2A-depleted breast cancer cells could be fully rescued by ectopic expression of JAG1 suggesting the involvement of NOTCH signaling in the regulation of SOX2 transcription.Previously, a genome-wide analysis of in vivo Rbpj targets in the neural stem cells also revealed that SOX2 is a potential target of NOTCH signaling [31].In addition, JAG1 has been demonstrated to increased SOX2 expression in the mammalian inner ear [32,33].However, whether NOTCH activation could stimulate SOX2 in cancer cells is still unknown.We identified three RBPJ binding sequences within the proximal human SOX2 promoter and demonstrated that NICD specifically binds to the Rbpj site located at the -1797/-1786 region of SOX2 promoter.Mutation of this site significantly attenuated JAG1-induced SOX2 promoter activity (Figure 5D and5E).However, this site is not participated in the regulation of basal SOX2 transcription.Our results confirmed SOX2 is also a direct transcriptional target of the JAG1/NOTCH signaling pathway in cancer cells. In addition to increase of stemness, we also demonstrated the promotion of tumor angiogenesis by KDM2A via PDGFA and JAG1.PDGFA is a well-characterized pro-angiogenic factor in different physiological and pathological conditions including organ development, ischemia and renal diseases [34][35][36].In addition, expression of PDGFA has been shown to be associated with high vascular density, lymph node metastasis and tumor recurrence in breast cancer [37,38].Our data indicated that PDGFA is a direct target of KDM2A and a critical mediator of KDM2Ainduced tube formation of endothelial cells (Figure 6).The angiogenesis-promoting activity of JAG1 has been elegantly demonstrated by showing that JAG1-expressing head and neck cancer cells triggered NOTCH activation in co-cultured endothelial cells and promoted capillarylike sprout formation in vitro and angiogenesis in vivo [39].In consistent with their results, we also found that JAG1-expressing breast cancer cells activated the NOTCH signaling and increased tube formation in endothelial cells.Knockdown of KDM2A in breast cancer abolished these stimulatory effects which could be reversed by JAG1 overexpression indicating JAG1 is involved in KDM2Ainduced tumor angiogenesis. Results of this study suggest KDM2A is a potential therapeutic target for breast cancer.Daminozide, a plant growth regulator, is the first KDM inhibitor selective for the KDM2/7 subfamily and inhibits KDM2A with an IC50 value of 1.5 μM [40].Suzuki et al developed a series of hydroxamate analogues and identified compound 9 as a potent inhibitor of KDM2A, KDM7A and KDM7B [41].Optimisation of triazolopyridine compounds yielded another selective KDM2A inhibitor, compound 35, which exhibits >30X selectivity over members of the KDM3, 4 and 6 subfamilies [42].Very recently, a novel cell-active KDM2A inhibitor was identified from high throughput screening [43].The antitumor activity of these compounds on orthotopic breast cancer models warrants further investigation. In contrast to our findings, Zizwani et al suggested a tumor suppressive role of KDM2A in breast cancer [44].Several discrepancies are discussed as following.First, the authors showed that KDM2A was stained in the myoepithelial cells while we found that KDM2A was expressed in cancer cells.When our manuscript was submitted for review, a new paper published by Tanaka et al demonstrated that KDM2A staining was detected in tumor and nontumor areas of breast cancer [45].As shown in their figure, the KDM2A signal in tumor part was appeared in cancer cells but not myoepithelial cells.Second, similar to our results, Tanaka et al concluded that the expression of KDM2A in breast cancer remained high during carcinogenesis while Zizwani et al concluded that KDM2A expression decreases with disease progression to metastasis.Among these three studies, only our study provided clear clinicopathological association in a large cohort of patients.Therefore, we think our study is of clinical significance.More importantly, analysis of the association of KDM2A with survival of breast cancer patients in two public databases that included more than 3,500 patients also indicated that high expression of KDM2A is linked with poor survival (Figure 1D and1E).Third, our and Zizwani's studies all pointed out the alteration of cell cycle progression after KDM2A depletion.However, the effect is very different.The discrepancy could be attributed to the knockdown strategy.In our study, we used stable clones established by antibiotic selection.The expression of KDM2A was continuously inhibited due to the constitutive expression of shRNA from the expression vector.Conversely, Zizwani et al transiently transfected siRNA to repress KDM2A in breast cancer cells.The efficiency of transient transfection may be different from assay to assay.In addition, the inhibition of KDM2A by siRNA will decrease after cell division because of the dilution of siRNA in the divided cells.More works are needed to clarify this issue. Taken together, we conclude that KDM2A functions as an oncogene in breast cancer by upregulating JAG1 to promote stemness, chemoresistance and angiogenesis. ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "MDA-MB-231 cell line purchased from the Bioresource Collection and Research Center (Taiwan) was cultured in RPMI1640 medium containing 10% fetal bovine serum (FCS).EA.hy926 cell line was kindly provided by Dr. Ming-Hing Tai (National Sun Yat-sen University, Taiwan).pLKO.shRNA-KDM2Aplasmid was obtained from the National Core Facility for Manipulation of Gene Function by RNAi, miRNA, miRNA sponges, and CRISPR/Genomic Research Center (Academia Sinica, Taipei).KDM2A expression vector was obtained from Dr. Yi Zhang (Harvard University).Daminozide was purchased from Cayman (Ann Arbor, Mi, USA).Anti-KDM2A and anti-SOX2 antibodies were obtained from Abcam (Cambridge, MA, USA).Anti-JAG1 and anti-PDGFA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).Anti-CD44 and anti-α-Tubulin antibodies were purchased from GeneTex Inc. (Hsinchu, Taiwan).Anti-CD24 antibody was obtained from StemCell Tecnologies Inc. (Vancouver, BC, Canada).Anti-cleaved Notch 1 (V1744) antibody was purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). ", "section_name": "Cell culture and reagent", "section_num": null }, { "section_content": "pLKO.shRNA-KDM2A plasmid was transfected into MDA-MB-231 cells with GeneIn transfection reagent (Amsbio, Cambridge, MA, USA).After transfection, the cells were cultured at 37°C in a 5% CO2-humidified atmosphere for 48 h and then subjected to antibiotic selection with 10 μg/ml puromycin.KDM2A expression of knockdown cells was detected by Western blotting and two stable clones (named as KDM-MB-231-2A2 and MDA-MB-231-3A1) were used in this study. ", "section_name": "Establishment of KDM2A knockdown stable cell line", "section_num": null }, { "section_content": "Cells (5000/well) were seeded onto 96 wells plates.After different times, 5 μg/ml of MTT reagent was added to each well and incubated for another 4 h.The reaction was stopped with 100 μl DMSO and the absorbance was detected at 570 nm using a microplate reader. ", "section_name": "Cell proliferation assay", "section_num": null }, { "section_content": "Total RNAs were isolated from MDA-MB-231 cells and two KDM2A-depleted stable clones by using an RNA extraction kit (Geneaid, New Taipei City, Taiwan).RNA samples were subjected to microarray analysis by Human OneArray v6 (Phalanx Biotech, Hsinchu, Taiwan).Data were analyzed with Rosetta Resolver System software (Rosetta Biosoftware, USA).Standard selection criteria to identify differentially expressed genes are as follows: (1) log 2 fold change ≥ 1 and P < 0.05.(2) log 2 ratios=\"NA\" and the differences of intensity between the two samples≥1000.Pathway analysis was analyzed with GSEA software. ", "section_name": "Human gene expression analysis", "section_num": null }, { "section_content": "Total RNA was isolated from cells and 1 μg of RNA was reverse transcriped to cDNA.Target mRNAs were quantified using real-time PCR reactions with SYBR green fluorescein and actin was served as an internal control.cDNA synthesis was performed at 95°C for 5 min, and the conditions for PCR were 30 cycles of denaturation (95°C/45 sec), annealing (60°C/45 sec), extension (72°C/45 sec), and 1 cycle of final extension (72°C/10 min).The primers used in this study were listed in Supplementary materials. ", "section_name": "Quantitative reverse transcription-PCR analysis (qRT-PCR)", "section_num": null }, { "section_content": "Cellular proteins were extracted from MDA-MB-231 or MDA-MB-231-2A2 cells with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM EDTA and 50 mM NaF) and the proteins were separated by SDS-PAGE.Proteins were transferred to PVDF membranes and the membranes were probed with various primary antibodies and developed by enhanced chemiluminescence reagent. ", "section_name": "Western blotting", "section_num": null }, { "section_content": "pGL3-Basic-SOX2 promoter vectors (0.5μg) was transfected into cells with GeneIn reagent in 24 well plate.After 24 h, cells were collected and lysed with 50 μL of freshly diluted reporter lysis buffer (Promega).After centrifugation, 20 μL of supernatant was added to 50 μL of the luciferase assay substrate (Promega) and the luminescence of the samples were read immediately by CentroLIApc LB 962 Microplate Luminometer (BERTHOLD TECHNOLOGIES GmbH & Co. KG, Germany), in which light production was measured for 1 second. ", "section_name": "Luciferase activity assay", "section_num": null }, { "section_content": "Cells were fixed with 1% formaldehyde at 37°C for 10 min and subsequently washed twice with ice-cold PBS containing protease inhibitors (1 mM phenylmethylsulphonyl fluoride, 1 μg/mL aprotinin, and 1 μg/mL pepstatin A).Cells were incubated in a lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) for 10 min on ice and sonicated to shear genomic DNA.The lysate was centrifuged for 10 min at 13000 rpm at 4°C.The supernatant was diluted in a ChIP dilution buffer (0.01% SDS, 1% Triton X-100, 2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, and protease inhibitors).Anti-KDM2A, anti-dimethyl H3K36, anti-trimethyl H3K36, anti-trimethyl H3K4 or non-immune (negative control) antibodies were added to the supernatant and incubated overnight at 4°C with rotation.DNA fragments were recovered and subjected to PCR amplification using specific primers for the detection of the CpG islands upstream of JAG1 or PDGFA gene transcription start site. ", "section_name": "Chromatin immunoprecipitation (ChIP) assay", "section_num": null }, { "section_content": "PKH67-stained EA.hy926 endothelial cells were incubated with MDA-MB-231, MDA-MB-231-2A2, MDA-MB-231-2A2-JAG1 or MDA-MB-231-2A2-PDGFA cells for 8 h in 24-well plates coated with 250 μl of 10 mg/ml Matrigel (BD).The total branching length and segments length were calculated by ImageJ software. ", "section_name": "Tube formation assay", "section_num": null }, { "section_content": "Cells were cultured with DMEM/F12 medium containing B27 supplement (GIBCO), 20 ng/ml EGF and 20 ng/ml FGF2 in low-attachment 6-well plates.After 7 or 14 days, spheres were collected without pipetting and fixed with 3.7% formaldehyde.The spheres with a diameter greater than 60 μm were counted.For the second sphere formation, the spheres collected at day 14 were dispersed to single cell by continuous pipetting and then re-seeded into the low attachment plate for another sphere formation assay. ", "section_name": "Sphere formation assay", "section_num": null }, { "section_content": "Cells were cultured with DMEM/F12 medium containing B27 supplement, 20 ng/ml EGF and 20 ng/ml FGF2 in low-attachment plates for 14 days.Cells were harvested, and incubated with anti-CD24 and anti-CD44 antibodies for 1 h at 4°C.Cells were washed and incubated with Alexa Fluor 594 anti-rabbit and Alexa Fluor 488 antimouse IgG antibody for another 1 h.The CD44 hi /CD24 - cells were detected by flow cytometry. ", "section_name": "Flow cytometric analysis of CD44 hi /CD24 -cancer stem cell population", "section_num": null }, { "section_content": "To test whether KDM2A inhibition could enhance chemosensitivity, the mammospheres generated from MDA-MB-231 cells were cultured in the absence or presence of 5μM Daminozide for 7 days in low-attachment plates.The mammospheres were collected and dispersed to single cells by continuous pipetting.The cells were seeded onto 24 well plates.After attachment, cells were treated with different concentrations of cisplatin for 24 h and viable cells were counted by using trypan blue exclusion assay. ", "section_name": "Cytotoxicity on tumor spheres", "section_num": null }, { "section_content": "MDA-MB-231 or MDA-MB-231-2A2 cells (1x 10 6 /mouse) were suspended in Hank's balanced salt solution and inoculated into the fourth mammary fat pads of 6 week-old female BALB/cAnN.Cg-Foxn1nu/ CrlNarl mice.After 4 weeks, tumor-bearing animals were sacrificed and the tumors were isolated from mice.The tumor weight was measured and the statistical difference between experimental groups was evaluated by t-test.Tumor tissues were used for immunohistochemical study.Animal use protocol was approved by the Institutional Animal Care and Use Committee of National Health Research Institutes. ", "section_name": "In vivo orthotopic animal study", "section_num": null }, { "section_content": "Paraffin-embedded tissue sections of human breast cancer specimens were obtained from Chi-Mei Medical Center (Tainan, Taiwan).The slides were stained with anti-KDM2A antibody and the signal intensity was interpreted using the H-score, defined by the following equation: H-score = ΣPi (i + 1) as previously described [46], where is the intensity of the stained tumor cells (0 to 3+), and Pi is the percentage of stained tumor cells with various intensities.Tumors with H-scores greater than the median of all cases were regarded as high expression.Survival analyses for disease-specific and metastasis-free survival were performed using Kaplan-Meier plots and compared using the log-rank test.This study was approved by the Research Ethics Committee of National Health Research Institutes.Written informed consent was obtained from all patients participated in this study. ", "section_name": "Immunohistochemical study of clinical samples", "section_num": null }, { "section_content": "Student t test was used to compare independent groups in our in vitro and in vivo experiments.Diseasespecific and metastasis-free survival were performed using Kaplan-Meier plots and compared using the log-rank test.Two-tailed P values ≤ 0.05 were considered statistically significant.Statistical analysis was performed using the GraphPad Prism version 5.01 (GraphPad Software, Inc.). ", "section_name": "Statistical analysis", "section_num": null } ]	[ { "section_content": "The authors thank Dr. Ming-Hong Tai and Dr. Yi Zhang for providing the materials.This work was supported by the grants MOHW104-TDU-B-212-124-003 and MOHW105-TDU-B-212-134007 of the Ministry of Health and Welfare and MOST-104-2320-B-400-027 of the Ministry of Science and Technology. ", "section_name": "ACKNOWLEDGMENTS", "section_num": null }, { "section_content": "The authors declared no competing financial interest. ", "section_name": "CONFLICTS OF INTEREST", "section_num": null }, { "section_content": "", "section_name": "Author's contributions", "section_num": null } ]
10.3389/fimmu.2022.781364	A Specific CD44lo CD25lo Subpopulation of Regulatory T Cells Inhibits Anti-Leukemic Immune Response and Promotes the Progression in a Mouse Model of Chronic Lymphocytic Leukemia	<jats:p>Regulatory T cells (Tregs) are capable of inhibiting the proliferation, activation and function of T cells and play an important role in impeding the immune response to cancer. In chronic lymphocytic leukemia (CLL) a dysfunctional immune response and elevated percentage of effector-like phenotype Tregs have been described. In this study, using the Eµ-TCL1 mouse model of CLL, we evaluated the changes in the Tregs phenotype and their expansion at different stages of leukemia progression. Importantly, we show that Tregs depletion in DEREG mice triggered the expansion of new anti-leukemic cytotoxic T cell clones leading to leukemia eradication. In TCL1 leukemia-bearing mice we identified and characterized a specific Tregs subpopulation, the phenotype of which suggests its role in the formation of an immunosuppressive microenvironment, supportive for leukemia survival and proliferation. This observation was also confirmed by the gene expression profile analysis of these TCL1-specific Tregs. The obtained data on Tregs are consistent with those described so far, however, above all show that the changes in the Tregs phenotype described in CLL result from the formation of a specific, described in this study Tregs subpopulation. In addition, functional tests revealed the ability of Tregs to inhibit T cells that recognize model antigens expressed by leukemic cells. Moreover, inhibition of Tregs with a MALT1 inhibitor provided a therapeutic benefit, both as monotherapy and also when combined with an immune checkpoint inhibitor. Altogether, activation of Tregs appears to be crucial for CLL progression.</jats:p>	[ { "section_content": "Despite the extensive research and the development of new treatment modalities, the number of chronic lymphocytic leukemia (CLL) cases with clinical resistance to therapy is constantly rising (1).The newest achievement in immunotherapychimeric antigen receptor T cells (CAR-T cells)are less effective in CLL as compared to other B cell malignancies, including B cell acute lymphoblastic leukemia or diffuse large B cell lymphoma (2)(3)(4).Similarly, immune checkpoint inhibitors have a limited efficacy in relapsed/ refractory CLL (3).In preclinical studies, antibodies against lymphocyte activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) are only effective when administrated in the initial stage of leukemia development (5)(6)(7).Importantly, the immune system dysfunctions observed in CLL patients, suggest that CLL cells modulate the microenvironment to their own benefit (8)(9)(10).The exhausted phenotype of T cells that display high expression of PD-1, LAG-3, or T cell immunoglobulin domain and mucin domain (TIM-3) is a hallmark of CLL (11,12).In order to improve the therapeutic strategies for CLL, it is crucial to understand the mechanisms that shape the leukemia microenvironment. Naturally occurring, thymic, Forkhead box protein P3 (FoxP3) + , CD4 + regulatory T cells (Tregs), are sensitive to activation by self-antigens and tumor neoantigens, and are main players of the neoplastic microenvironment (13).Tregs can affect T cells in all stages of immune response development: priming, proliferation, and T cell effector functions (14).Increased frequency of Tregs correlates with poor prognosis of CLL patients (15).The expression patterns of Tregs-associated markers (CD25, LAG-3, killer cell lectin like receptor G1, CD69, Eomesodermin -EOMES) that determines their suppressive functions was recently presented in both CLL patients and leukemia-bearing mice (5,16,17).Nevertheless, the function of Tregs in CLL has not been elucidated and the approaches for Tregs elimination have shown to be insufficient.For instance, the administration of anti-CD25 antibodies or phosphoinositide 3kinase d (PI3Kd) inhibitors affected not only Tregs but also abrogated the activation and function of CD8 + lymphocytes (18). In order to evaluate the role of Tregs in the development and shaping of immunosuppressive microenvironment of CLL, in this work we used Eµ-TCL1 transgenic mice model (19,20).We characterized a novel, TCL1-derived Tregs subpopulation and assessed Tregs suppressive activity in functional tests.Furthermore, TCR sequencing allowed us to better understand the influence of leukemia on Tregs and CD8 + T lymphocytes activation and clonality.Finally, we used the inhibitor of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) to block the activation of Tregs.MALT1 protease is a component of CARMA1-BCL10-MALT1 (CBM) complex which was shown to be crucial for Tregs activity (21).The results obtained in this study provide the evidence that Tregs are essential for leukemia progression in immunocompetent mice and can be efficiently targeted to block CLL progression. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "MI-2 (Malt1 inhibitor, Selleckchem.com)was dissolved in DMSO (Sigma-Aldrich, St Louis, MA, USA), aliquoted and stored at -20°C.Albumin from chicken egg white (OVA, Sigma-Aldrich, St Louis, MA, USA) and Poly (I:C) (HMW) (In vivoGen, San Diego, CA, USA) were aliquoted and stored at -20°C.Diphtheria Toxin (DT) from Corynebacterium diphtheriae (Sigma-Aldrich, St Louis, MA, USA) was aliquoted and stored at -80°C.Anti-mouse PD-L1 antibody InVivoPlus (B7-H1) (BioXcell, Lebanon, NH, USA) and InVivoPlus rat IgG2b isotype control, (BioXcell, Lebanon, NH, USA) were stored at 4°C. ", "section_name": "Reagents", "section_num": null }, { "section_content": "All in vivo studies were performed in accordance with the EU Directive 2010/63/EU and the Polish legislation for animal experiments of the Polish Ministry of Science and Higher Education (February 26, 2015) and approved by the Local Ethics Committee for the Animal Experimentation in Warsaw.The in vivo experiments were carried out in Animal Facility of the Medical University of Warsaw. For the study, 6-12 weeks old female or male (never mixed in one experiment) mice were used.Mouse strains include: C57BL6/J (wild-type, immunocompetent mice) (Medical University of Bialystok or Mossakowski Medical Research Centre), B6.Cg-Foxp3tm2(EGFP)Tch/J (B6 Foxp3 EGFP , Tregs express GFP) (University of Warsaw), C57BL/6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax (DEREG, Tregs express GFP and receptor for diphtheria toxin) (The International Centre for Genetic Engineering and Biotechnology, Trieste, Italy) B6(Cg)-Rag2tm1.1Cgn/J(RAG2-KO, immunodeficient mice) and C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-1) (Medical University of Warsaw).Splenocytes or leukemic CD5 + CD19 + TCL1 cells (5×10 6 -1×10 7 ) isolated from spleens of female Em-TCL1 transgenic mice (The International Centre for Genetic Engineering and Biotechnology, Trieste, Italy) were adoptively transferred via tail vein injection.In described experiments we used cells isolated from two different Eµ-TCL1 transgenic mice, either TCL1-1159 or TCL1-1013.These cells were propagated in mice maximally twice, with the exception of genetically modified TCL1 cells expressing OVA (due to the procedure of generating modified cells, they required additional propagation in RAG2-KO mice). ", "section_name": "Animals Studies", "section_num": null }, { "section_content": "Eµ-TCL1 mice model of CLL was used in this study.To monitor leukemia development and progression, the percentage of leukemic TCL1 cells (CD5 + CD19 + ) among white blood cells (WBC) in the peripheral blood (PB) collected from cheek vein was assessed by flow cytometry.The consistency in the assessment of leukemia was ensured and blinding practice was not applicable.Mice with detected leukemia were randomly selected and further used in the experiments.The sample size was calculated with power analysis test (22). DEREG mice were treated with DT (50 µg/kg) administered intraperitoneally (i.p.) every four days.MI-2 was administered i.p. daily, at dose 20 mg/kg and the control mice were injected with the DMSO as a solvent.Anti-PD-L1 antibody or the appropriate isotype control were administered i.p. every second day at a dose 200 mg/mouse.The schemes of the treatments are presented in details in the appropriate figures. ", "section_name": "In Vivo Treatments", "section_num": null }, { "section_content": "In order to prepare a single cell suspension, spleens (SPL) or lymph nodes (LNs) were cut in small pieces and passed through a 150 mm cell strainer.To remove red blood cells the isolated splenocytes were lysed with ACK Lysing Buffer (Thermofisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.CD19 + , CD4 + and CD8 + cell subpopulations were isolated by immunomagnetic negative selection using EasySep ™ Mouse B Cell Isolation Kit, EasySep ™ Mouse CD4 + T cell Isolation Kit and EasySep ™ Mouse CD8 + Cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada), respectively, according to the manufacturer's protocols.The efficacy of the isolation was over 90%. ", "section_name": "Cell Isolation", "section_num": null }, { "section_content": "CD8 + cells isolated from spleens were incubated with CellTrace ™ Violet Cell Proliferation kit (CT) (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at 37°C, washed with cell culture medium and seeded onto 96-well U-bottom plates coated with anti-CD3 antibody (eBioscience, San Diego, CA, USA) together with sorted Tregs-GFP (either all GFP + or GFP + CD69 high CD44 -/low ) in various ratios (1:0.125,1:0.25, 1:0.5, 1:1 and 1:2).For stimulation, anti-CD28 (eBioscience, San Diego, CA, USA) antibody was added to the culture medium.The proliferation of CD8 + cells was evaluated upon 72h using BD FACSCanto ™ II Flow Cytometer and BD FACSDiva Software (v8.0.1) (BD Biosciences, La Jolla, CA, USA). ", "section_name": "CD8 + Cells Proliferation Assay", "section_num": null }, { "section_content": "The sequence encoding ovalbumin (Addgene, cat.number 25097) was inserted into mammalian expression vector pCDH-EF1-MCS-T2A-copGFP (System Biosciences).The pCDH-EF1-OVA-GFP and a packaging (psPAX2) and an envelope (pMD2.G) plasmids (gifts from prof.Didier Trono, E ́cole Polytechnique Fedeŕale de Lausanne, Switzerland) were introduced into HEK-293T cells using Polyethylenimine (Polysciences).Then freshly isolated TCL1 cells (CD5 + CD19 + ) from mouse spleens were seeded into 24-well plates with M2-10B4 murine stroma cells.Next, medium containing lentiviral particles was added into TCL1 and M2-10B4 cells co-culture.Then TCL1 cells were washed and inoculated into RAG2-KO mice for leukemic cells propagation.Finally, OVA + GFP + cells were sorted and used for further experiments.In all performed experiments at least 60% of injected leukemic cells exerted OVA + GFP + phenotype as evaluated by flow cytometry. ", "section_name": "TCL1 OVA-Expressing Cells", "section_num": null }, { "section_content": "Two weeks following TCL1 cells adoptive transfer, leukemiabearing DEREG transgenic mice were treated with DT and on the following day, injected with CT-positive CD8 + T cells isolated from spleens and lymph nodes of OT-1 mice.4-5 hours later, the mice were i.v.inoculated with OVA protein (50 µg).The proliferation of CD8 + OT-1 cells isolated from spleens was assessed upon 3 days using flow cytometry.In the second approach, DEREG mice were injected with genetically modified TCL1 leukemic cells expressing OVA-GFP (TCL1-OVA).Three days later, the mice were treated with DT and on the following day, injected with CT-positive CD8 + OT-1 T cells.The proliferation of CD8 + OT-1 cells was evaluated following 3 or 4 days using flow cytometry.The schemes of described experiments are presented in detail on appropriate figures. ", "section_name": "In Vivo Functional Assays", "section_num": null }, { "section_content": "The isolated cells were stained with Zombie NIR ™ Fixable Viability kit or Zombie Violet ™ Fixable Viability Kit (BioLegend, San Diego, CA, USA) for 20 min at room temperature (RT) and washed with PBS.Next, the cells were incubated with Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block ™ ; clone 2.4G2, BD Biosciences, La Jolla, CA, USA) for 15 min at RT and stained for surface markers with proper fluorochrome-conjugated antibodies (all antibodies used in this study are listed in Supplementary Table 1) for 20-30 min at RT.After final washing with PBS, the cells were analysed using BD FACS Canto ™ II Flow Cytometer and BD FACS Diva Software (v8.0.1)(BDBiosciences, La Jolla, CA, USA).For further analyses, including t-SNE (with markers: CD44, CD25, LAG-3, CD69), FlowJo Software (v.10.6.1)(FlowJo LLC, Ashland, OR, USA) was used. ", "section_name": "Flow Cytometry", "section_num": null }, { "section_content": "In order to sort Tregs (CD4 + , GFP + ) from spleens of B6 Foxp3 EGFP or DEREG mice, CD4 + cell subpopulation was enriched prior to sorting.To this end, isolated splenocytes were subjected to immunomagnetic positive selection for CD19 + using EasySep ™ Mouse CD19 Positive Selection Kit II (STEMCELL Technologies) and then the negative fraction was subsequently subjected to negative selection using EasySep ™ Mouse CD4 + T cell Isolation Kit (STEMCELL Technologies).When needed, CD4 + cells were additionally stained with anti-CD69-PE and anti-CD44-PE-Cy7 monoclonal antibodies as described above.To sort CD8 + cells, the fraction of splenocytes devoid of CD19 + cells was stained with anti-CD8a-PerCP-Cy5.5 monoclonal antibody.Then the cells were sorted using BD FACS Aria ™ III Cell Sorter (BD Biosciences). ", "section_name": "Cell Sorting", "section_num": null }, { "section_content": "Tregs A (GFP + , CD69 high , CD44 -/low , gated as presented in Figure 5 CD69/CD44 right panel) and CD8 + cells were sorted as described above.Then the genomic DNA was isolated from the sorted cells using DNA Micro Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.The concentration and purity of extracted DNA was assessed using NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific).Immunosequencing of the CDR3 regions of TCRb chains was performed with immunoSEQ ® Assay and analysed by immunoSEQ ® Analyzer (Adaptive Biotechnologies, Seatlle, WA, USA). ", "section_name": "DNA Isolation and Analysis of TCRb Repertoire", "section_num": null }, { "section_content": "When percentage of leukemic cells in mouse blood reached at least 20% of all PBMC, the GFP + Tregs: A (GFP + , CD69 high , CD44 -/low ) and B (GFP + excluding fraction A) were sorted from TCL1 leukemia-injected DEREG mice.Additionally, GFP + Tregs were also sorted from control DEREG mice.The mRNA was isolated from 4.5 × 10 5 cells with the RNeasy Micro Kit (Qiagen, Hilden, Germany).Libraries were prepared with the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen), according to manufacturer's instructions, with the addition of UMI.Barcoded samples were pooled, diluted, loaded onto a NextSeq 500/550 Mid Output flowcell (130M reads, Illumina) and single-end 150bp sequencing was performed on a NextSeq 550 (Illumina). After initial QCs using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FastQ Screen (https:// www.bioinformatics.babraham.ac.uk/projects/fastq_screen/), fastq files were processed using a local Snakemake workflow including the following main steps.First, raw reads were trimmed from their UMI index, poly A and adapter sequences using a combination of dedicated scripts and cutadapt (v2.10).Next, filtered reads were submitted for mapping (STAR v2.5.3a) on the Mouse Reference genome (GRCm38).Collapsing of reads originating from the same fragment was achieved with umi_tools (v 1.0.0) and counting was performed with featureCounts (subread v2.0.0). Counts were filtered and transformed with edgeR (cpm > 5 and presence in at least 3 samples).For data visualization, heatmaps, sample distance matrix, and volcano plots were drawn with EdgeR, heatmap, and EnhancedVolcano R packages.For differential expression of genes across samples (DEGs), FDR < 0.05 and log2 fold change cut-off of 1 were imposed.For clustering, DEGs were selected as important for immune functions in Tregs.Gene expression values were z-scored and subjected to correlation-based clustering with complete linkage.Raw and processed data were deposited at the NCBI GEO database (GSE179121).The following secure token has been created to allow review of record GSE179121 while it remains in private status: qlchuysazpinjed.To better understand the nature of Tregs A and Tregs B, we re-analyzed the public dataset GSE72494 describing the transcriptome of naive, activated, and effector Treg (23) and performed a Gene Set Enrichment Analysis (GSEA, Hallmark and curated gene sets) with the stand-alone software (GSEA v4.2.1, Broad Institute, Boston, MA).Normalized enrichment scores (NES) and p-values < 0.05 were taken into consideration. ", "section_name": "RNA Sequencing", "section_num": null }, { "section_content": "GraphPad Prism 6 Software (GraphPad Software Inc., San Diego, CA, USA) was used for data analysis.The statistical significance was calculated by Mann-Whitney U test.The mice survival rate was analyzed by log-rank survival test.For gene expression data (RNA sequencing), one-way ANOVA with multiple comparisons was calculated for single genes and histograms were drawn with GraphPad Prism 9 Software.Additional experimental procedures are described in details in the Supplementary Material. ", "section_name": "Statistical Analysis", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "To evaluate the significance of Tregs for CLL progression we used DEREG transgenic mice with depletion of FoxP3 + CD4 + Tregs by treatment with diphtheria toxin (DT) (Figure 1 and Supplementary Figure 1A,B).DEREG mice were treated with DT one day prior to adoptive transfer of malignant (CD5 + CD19 + ) B cells, isolated from an Eµ-TCL1 transgenic mouse.Effective depletion of Tregs was observed in spleens and peripheral blood of DEREG mice and was maintained by additional DT injections every four days (Figure 1A and Supplementary Figure 1A).As monitored in peripheral blood twice a week, injection of DT did not affect the progression of leukemia during the first fifteen days of experiments.However, starting from day 18 th after TCL1 leukemia inoculation, we detected a significant decrease in the percentage of leukemic cells (CD5 + CD19 + ), in the peripheral blood, of DT-treated mice as compared to untreated TCL1 leukemia-bearing animals (Figure 1B, left panel).In line with these results, we observed a significant reduction of previously established leukemia in the spleens of Tregs-depleted mice (Figure 1B, right panel).The same observations were made when DEREG mice were injected with TCL1 leukemia isolated from another transgenic mouse (Supplementary Figure 1B).The decrease in the percentage of leukemic cells in spleens of DTtreated mice was accompanied by the extensive increase of the percentage in both CD4 + and CD8 + T lymphocytes (Figure 1C).These observations prompted us to investigate the putative changes in the phenotype of splenic CD4 + and CD8 + T cells mediated by Tregs-depletion.We observed the enrichment of effector (EFF; CD44 + CD62L -) and central memory (CM; CD44 + CD62L + ) cells in both CD4 + and CD8 + T cell subpopulations in mice deprived of Tregs (Figures 1D,E).A significant increase in the percentage of effector (CD4 + and CD8 + ) and central memory (CD8 + ) T cells was also observed in lymph nodes (axillary, brachial, inguinal) of DT-treated mice (Figure 1F).Depletion of Tregs resulted in the elevation of CD69 on both, CD4 + and CD8 + T cells in the lymph nodes, and reduced the percentage of naïve cells, suggesting the activation of a systemic immune response.Nevertheless, the depletion of Tregs, performed at an advanced stage of the disease (first dose of DT was administered when 30% of malignant B cells were detected among all white blood cells in peripheral blood) did not affect the progression of leukemia (Supplementary Figure 1C).Tregs depletion at an advanced stage of leukemia progression increased the percentage of effector and IFN-gpositive CD4 + and CD8 + T lymphocytes, significantly elevated IFN-g concentration and reduced the concentration of IL-10 in the sera (Supplementary Figure 1D,E).Importantly, three weeks of DT injections of control (without leukemia) DEREG mice lead to a minor activation of T cells, mostly CD4 + T cell subpopulation (Supplementary Figure 1F, upper panel).However, no changes were observed in the level of CD69 in lymphatic T cells upon DT-treatment (Supplementary Figure 1F, lower panel).The activation of CD4 + T cells may be the result of anti-DT immune response as it was described before (24). To understand more deeply the anti-leukemia immune response induced by Tregs depletion, we investigated the impact of CD8 + T lymphocytes derived from the mice after DT injections on leukemia progression.We limited these experiments to the subset of CD8 + T cells as it was shown that these cells play a superior role in anti-leukemia immune response over CD4 + T lymphocytes (25).Importantly, an effective Tregs depletion in DEREG mice is transient.At day 22 after TCL1 injection we observed that the Tregs population was restored in murine blood despite continuous injections of DT (Supplementary Figure 1A, right panel), as was also reported by others (26).Thus, to examine the impact of the CD8 + lymphocytes on leukemia progression and mice survival, the cells were isolated from spleens of untreated or DT-treated TCL1 leukemia-bearing mice (the same scheme of experiment as shown in Figure 1A), and adoptively transferred into TCL1-injected RAG2-KO mice (Figure 2A).Next, the expansion of TCL1 leukemic cells was monitored in murine blood twice a week.Interestingly, CD8 + T lymphocytes, isolated from Tregs-depleted mice effectively prevented leukemia progression, and in some mice even lead to complete elimination of TCL1 cells (Figures 2B,C).In contrast, the CD8 + T lymphocytes adoptively transferred from mice with intact Tregs population did not significantly affect the progression of the disease in RAG2-KO mice.Consequently, in TCL1-injected RAG2-KO mice, the adoptive transfer of CD8 + T cells isolated from DEREG mice after Tregs depletion, translated into prolonged survival and complete leukemia eradication in three out of nine mice.(Figure 2D). The results obtained from the experiments described above revealed that the lack of Tregs in the leukemia microenvironment triggers the expansion of anti-leukemic CD8 + T cells.To address the differences in the investigated T cells, the CD8 + T cells from spleens of TCL1-injected DEREG mice treated with DT or untreated were sorted for DNA isolation and the T cell receptor beta chain (TCRb) third complementarity-determining regions (CDR3) sequences analysis.An increase of CD8 + T cell clonality was observed in three out of five TCL1 leukemia-bearing mice with Tregs depletion, but overall, the observed differences were not statistically significant between the two examined groups (Figure 2E).Strikingly though, we observed distinct amino acid sequences of TCRb CDR3 regions in the tested CD8 + T cells, suggesting different specificity of the T cells among untreated and DT-treated mice (Figure 2F).Indeed, only one sequence is shared in the top fifteen rearrangements between both analyzed CD8 + T cell populations (Figure 2F).Altogether, these data indicate that the elimination of Tregs from the TCL1 leukemia microenvironment resulted in the expansion of a distinct set of cytotoxic CD8 + T effector cells, capable of clearing leukemia in DEREG and RAG2-KO mice. ", "section_name": "Depletion of Tregs in Mice With Adoptively Transferred TCL1 Leukemia Results in the Expansion of Functional CD8 + Cells and Leukemia Clearance", "section_num": null }, { "section_content": "We analyzed the phenotype and function of Tregs in TCL1injected B6 FoxP3 EGFP transgenic mice that express EGFP and FOXP3 under the control of endogenous promoter.Based on the results of phenotyping with a set of markers (FoxP3, LAG-3, CD69, CD44, CD25), we performed t-distributed stochastic neighbor embedding (tSNE) analysis, which allowed us to distinguish a specific Tregs subpopulation that exerts the phenotype characteristic only for Tregs isolated from TCL1 leukemia-bearing mice (Tregs A) (Figure 3A).This particular Tregs A subpopulation can be defined by high level of CD69, LAG-3 and low of CD44 and CD25 on their surface. In order to investigate whether the observed changes in Tregs phenotype are mediated by the interactions with malignant B cells, we co-cultured the control Tregs-GFP + (sorted from spleens of control B6 Foxp3 EGFP mice) with leukemic (TCL1) or normal (CD19) B cells.After three days, significantly higher level of LAG-3 was observed on Tregs-GFP + co-cultured with TCL1 cells, but not with the control CD19 + cells (Supplementary Figure 2A).The elevated expression of LAG-3 was achieved only when Tregs-GFP+ and TCL1 leukemia cells were cultured in direct contact.On contrary, the level of CD44 in Tregs-GFP + co-cultured with TCL1 leukemia cells (but not normal CD19 + cells) was reduced regardless the separation of the cells by transwells (Supplementary Figure 2B). Next, the clonality of Tregs A subpopulation was examined, based on the TCRb CDR3 region sequences.Importantly, the TCL1-associated Tregs A subpopulation sorted from spleens of TCL1-leukemia bearing DEREG mice exhibits increased clonality and elevated frequency of particular clones, compared to whole Treg-GFP + subpopulation sorted from the control animals (CTR Tregs) (Figures 3B,C). Finally, in order to characterize the TCL1-associated Tregs at the transcriptomic level, we performed RNA sequencing on two subpopulations of Tregs sorted from the spleens of TCL1-injected DEREG mice: Tregs A (specific to Eµ-TCL1 model, sorted as GFP + CD44 -/lo and CD69 hi ) and Tregs B (the remaining GFP + Tregs, which did not meet the criteria of subpopulation A).The transcriptome of both TCL1-associated subpopulations was compared with Tregs-GFP + population sorted from spleens of control mice.Interestingly, the analysis of differentially expressed genes (DEGs), showed that Tregs A subpopulation was markedly different from both Tregs B as well as control Tregs populations (Supplementary Figure 3,4).This data suggests that the specific Tregs A cells signature might be selectively induced within the conditions of leukemia progression.In Tregs A, we observed increased expression of genes responsible for immunosuppressive activity (Gzmb, Prf1, Gzmk, Il10), checkpoints (Havcr 2, Lag-3, Tigit), chemokines that may support leukemia progression and its microenvironment (Ccl3, Csf1, Ccl5), as well as genes that have been recently reported as unique for CLL-Tregs (EOMES) (Figure 3D).Importantly, the gene expression profile was in line with the phenotype observed in flow cytometry, apart from CD69, which seemed to be regulated post-transcriptionally (Figure 3E).Additionally, the elevated level of Ikzf2 encoding Helios transcription factor suggests enhanced suppressive capacity of Tregs A subpopulation (27). Next, we compared the gene expression profiles of Tregs A and B with a public dataset [GSE72494 (23)] describing the transcription profiles of naive, activated, and effector Tregs.We used a gene signature reported in that study (23), and built heat maps to 2 and3). Although Tregs A exhibited comparable transcription changes as compared to Effector Tregs (up-regulation of Il10 and Havcr2/Tim3 and down-regulation of Sell and Ccr7), we identified important differences suggesting a particular gene modulation in this specific Tregs population found in CLL (e.g.Eomes, Prf1, Itgae, Cxcl10) (Supplementary Figure 4D). ", "section_name": "CLL Leads to the Formation of a Specific Population of Tregs", "section_num": null }, { "section_content": "Next, we determined the ability of splenic Tregs population, sorted from control and TCL1 leukemia-bearing B6 FoxP3 EGFP mice to inhibit CD8 + T cell proliferation in an antigen unspecific test, where T cells were activated via anti-CD3 and anti-CD28 antibodies.The obtained results indicated that whole Tregs population isolated from spleens of TCL1 leukemia-bearing mice is prone to inhibit CD8 + T cells proliferation similarly to control Tregs (Figure 4A).Similarly, in a test with OVA peptide presented by the bone marrow-derived dendritic cells, Tregs isolated from leukemic mice inhibited CD8 + OT1 cells proliferation as effectively as Tregs from control mice (Supplementary Figure 5).This data suggest that the effectiveness of antigen-independent suppression of Tregs from TCL leukemia-baring mice is similar to control Tregs.In order to explore whether TCL1-associated Tregs suppress CD8 + T cells in an antigen specific manner, we generated OVAexpressing TCL1 by means of lentiviral transduction.DEREG mice were inoculated with TCL1-OVA cells, and 3 days later, Tregs were depleted with DT in one group.On the following day, mice were injected with Cell Trace (CT)-positive OT1 CD8 + T cells and the proliferation of these cells in the spleen was subsequently analyzed (Figures 4B-D).Interestingly, although the T cells were effectively activated in all tested TCL1 leukemia-bearing mice, in the group treated with DT, the proliferation was more efficient, suggesting that the Tregs population impeded OT1 CD8 + T cells proliferation to some extent.Moreover, a significant drop in the percentage of leukemic cells in blood and spleens of DT-treated mice was observed after injection of OT1 CD8 + T lymphocytes (Figure 4B).Conversely, when mice were inoculated with TCL1 cells (without OVA expression) and subsequently injected with OVA protein, no impact of Tregs depletion was observed on OT1 CD8 + T cells proliferation (Figure 4C).Altogether, these results suggest that Tregs inhibit proliferation of leukemia-specific CD8 + T cells in an antigen-dependent manner. ", "section_name": "The TCL1-Induced Tregs Are Functionally Active", "section_num": null }, { "section_content": "The analysis of Tregs phenotype at the various stages of leukemia revealed significant changes in the expression levels of Tregs surface proteins.The shift of Tregs into Tregs A phenotype escalated during leukemia progression and was accompanied by an increase in the percentage of splenic Tregs in leukemic mice (Figure 5A).Importantly, the Tregs A subpopulation was clearly formed at an advanced stage of the disease (when more than 40% of leukemic cells among all white blood cells were present in the spleens). MI-2 has been described as a para-caspase MALT1 inhibitor that can selectively prevent the conversion of naïve Tregs into effector cells by decreasing the NFкB activity (21).MI-2 revealed its cytotoxic effect on primary CLL cells in vitro (28).Moreover, RNA sequencing analysis indicated elevated expression of NFкB-related genes in Tregs of TCL1-injected mice (Supplementary Figure 4B).In order to verify the influence of MI-2 on development of Tregs subpopulations, the inhibitor was administered intraperitoneally to the control and TCL1 leukemia-bearing B6 FoxP3 EGFP mice for two weeks starting from day 5 following TCL1 leukemic cells inoculation (Figure 5B).Administration of MI-2 impeded the change of Tregs into Tregs A phenotype and elevated the percentage of naïve Tregs (CD62L + CD44 -) (Figure 5C).MI-2 inhibited the progression of leukemia and increased significantly the percentage of central memory and effector CD4 + and CD8 + T lymphocytes (Figures 5C-E).Importantly, the effectiveness of MI-2 treatment was impaired in TCL1 leukemia-bearing RAG2-KO mice as compared to immunocompetent, wild type mice, suggesting a key role of T cells in the mechanism of action of this drug (Supplementary Figures 6A,B). Since the PD1/PD-L1 axis was already shown to contribute to T cells dysregulations in both human and mouse models of CLL, we used MI-2 therapy as a pretreatment for checkpoint blockade with anti-PD-L1 antibody in immunocompetent TCL1-leukemia bearing mice (6,29).Considering that long-term inhibition of Tregs functions can lead to autoimmune pathology (30), MI-2 inhibitor was used only before anti-PD-L1 therapy (Figure 6A).The anti-PD-L1 therapy did not affect the percentage of T cells already elevated by MI-2 (Figure 6B).However, the combined treatment decreased the percentage of naive cells and increased the percentage of effector cells of both CD4 + and CD8 + T lymphocytes (Figure 6B) Anti-PD-L1 antibodies administered 16 days post TCL1 inoculation decreased the percentage of leukemic cells in blood and spleen when applied after treatment with MI-2 (Figure 6C).These results indicate that the combination of Tregs inhibition with anti-PD-L1 antibody can bring beneficial treatment outcome in leukemia. ", "section_name": "Treatment With MALT1 Inhibitor Disturbs the Formation of Tregs A Subpopulation in TCL1 Leukemia-Bearing Mice and Enhances the Effect of Immunotherapy", "section_num": null }, { "section_content": "The anti-tumor strategy reducing the number of Tregs has been reported since 1999 (31).Nevertheless, targeting Tregs can yield differential responses in cancer models (32).In this study, we revealed that in the CLL mouse model, the depletion of Tregs population can lead to the expansion of CD8 + T cells with the ability to completely eradicate leukemia. Published studies have consistently demonstrated elevated levels of Tregs in the peripheral blood collected from CLL patients compared to healthy subjects (33).The phenotype of analyzed Tregs was described as effector-like in both CLL patients and the Em-TCL1 mouse model of CLL (5,16,18).Our results indicate that the phenotype of Tregs changes during the course of leukemia to establish a subpopulation of CD4 + , FoxP3 + , LAG-3 + , CD69 hi , and surprisingly, CD44 lo and CD25 lo cells.A low expression of CD25 in Tregs has been already reported by another group (34), yet the CD44 lo phenotype is rather a characteristic feature of naïve lymphocytes.Our ex vivo experiments revealed that the level of cell-surface glycoprotein CD44 decreased in Tregs as a result of leukemia progression.At the transcriptomic level, however, the reduced amount of mRNA for CD44 was seen only in Tregs A, a specific TCL1-associated Tregs subpopulation distinguished for the first time in this study.Interestingly, the Tregs A subpopulation is positive for already reported markers of CLL-related Tregs, including IL-10, LAG-3, granzyme B, EOMES, as well as share a unique gene expression signature of chemokines that may support leukemia progression and formation of leukemic microenvironment (35,36).Moreover, the overexpression of mRNA encoding HELIOS, TIGIT, TIM-3 and CD27 suggests that TCL1-related Tregs may possess immunosuppressive activity (27,(37)(38)(39).Mpakou and colleagues show that Tregs isolated from CLL patients have an ability to inhibit CD8 + T cell proliferation (34).Likewise, according to our results, TCL1-derived Tregs are able to inhibit proliferation of T cells ex vivo.In ex vivo assays, T cells were activated in unspecific and specific manner, accordingly with the cognate antigen or by OVA peptide presented by dendritic cells thus the observed effect was not related to leukemia-specific antigens.The CLL-related Tregs functionality was finally confirmed in the in vivo experiment with TCL1-OVA cells, indicating that Tregs inhibit the proliferation of CD8 + cells upon recognition of tumor-expressed antigen. The variable CDR3 regions of TCR interact with the peptide presented by MHC.The analysis of CDR3 sequence provides information about the diversity and clonality of investigated T cell populations and has become a valuable research tool in immunology (40).Thus, the higher oligoclonal composition of TCL1-derived Tregs compared to Tregs sorted from control mice, suggests that only selected clones of Tregs have undergone the expansion in TCL1 leukemia-bearing mice. The expansion of exhausted T cells is a hallmark of human CLL and is also recapitulated in the Eµ-TCL1 mouse model (11).The CD8 + lymphocytes, which are present in spleens of TCL1 leukemia-bearing mice, have been described as antigenexperienced, oligoclonal cells that expand during the progression of the disease (12).In our experiments, upon depletion of Tregs, the CD8 + T cells became more oligoclonal and were effective in the elimination of leukemic cells.Surprisingly, anti-leukemic CD8 + T cells expressed different CDR3 sequences compared to the CDR3 sequences of lymphocytes from non-DT-treated, leukemia-bearing mice.It has been reported that in some tumors, based on the TCR sequences functional T cells formed a distinct group from dysfunctional, transitional tumor -infiltrating lymphocytes (41).Our results suggest that the depletion of Tregs in leukemia-bearing mice triggers the expansion of functional CD8 + T cell clones through the presentation of different epitopes than those used for splenic, exhausted CD8 + T cells.Elimination of Tregs primed the activation of T cells not only in spleens but also in the lymph nodes.The expansion of CD8 + T lymphocytes capable of killing leukemic cells occurred due to Tregs depletion, thus revealing their role in the maintenance of tumor antigen tolerance in CLL.The limitation of these studies is the fact that the antigens that led to the activation of antileukemic T lymphocytes were not identified yet.However, we suspect that these antigens could be associated with mutations typical for CLL.Importantly, we cannot rule out the possibility that these antigens are of other origins, for example derived due to genetic differences between mouse strains.Nevertheless, the depletion of Tregs seems to be a trigger for the expansion of effector T lymphocytes. As it was also shown for other malignances, the inhibition of Tregs activation must occur early in the course of the disease to bring the beneficial outcome (42,43).It has also been shown that the efficacy of adoptive T cell therapy is dependent on the tumor burden and is high in the early stages of tumor development or after chemotherapy (44,45).To address this observation we conducted treatment with the MALT1 inhibitor, MI-2, when the leukemic cells were already detectable in blood but at a low level.MI-2 disrupted Tregs activation, prevented the formation of the specific TCL1-derived Tregs A subpopulation and inhibited the progression of leukemia in immunocompetent mice.Since the MI-2 was shown to exert a cytotoxic effect on leukemic cells (28), it is difficult to conclude from our experiments, whether it affects Tregs directly or only delays their activation due to the inhibition of leukemia progression.Though, the relatively small anti-leukemia efficacy of MI-2 obtained in RAG2-KO mice model may bring to the conclusion that T cells are important component in anti-leukemic MI-2 mechanism of action.Moreover, the decrease in the frequency of activated Tregs provided the therapeutic window to reduce the percentage of leukemic cells in mouse blood even two weeks after inoculation of leukemic cells. Our results underline the role of Tregs in the progression of CLL and more importantly suggest that reactivation of the existing, exhausted T cell populations with anti-PD-L1 therapy, might be insufficient to block the disease progression.Notably, the presented results indicate that one approach to obtain an effective anti-leukemia immune response is to reorganize the CLL microenvironment, in order to create an opportunity for the expansion of a population of cytotoxic CD8 + T cells. ", "section_name": "DISCUSSION", "section_num": null } ]	[ { "section_content": "We wish to thank Ewa Kozlowska from University of Warsaw for providing Treg-Foxp3EGFP mice, Serena Zacchigna and Simone Vodret from ICGEB, Trieste for their support with DEREG mice experiments and sharing the DEREG colony, and Nathalie Nicot, Elise Mommaerts, Arnaud Muller (LUXGEN Platform, LIH) for RNA sequencing. ", "section_name": "ACKNOWLEDGMENTS", "section_num": null }, { "section_content": "This work was supported by: the Polish National Science Centre grants 2018/29/B/NZ6/01962 (AM) and 2016/21/B/NZ7/02041 (MF) and the Ministry of Science and Higher Education within \"Regional Initiative of Excellence\" program in the years 2019-2022 -013/RID/2018/19, the FNRS \"Teĺevie\" 7.6518.20 (GP), and the Fonds National de la Recherche Luxembourg TIME-CLL: C20/BM/14582635 (EM). ", "section_name": "FUNDING", "section_num": null }, { "section_content": "The datasets presented in this study can be found in online repositories.The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/,GSE179121. ", "section_name": "DATA AVAILABILITY STATEMENT", "section_num": null }, { "section_content": "The animal study was reviewed and approved by The Local Ethics Committee for the Animal Experimentation in Warsaw, Warsaw University of Life Sciences, Warsaw, Poland. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.781364/full#supplementary-material Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2022 Goral, Firczuk, Fidyt, Sledz, Simoncello, Siudakowska, Pagano, Moussay, Paggetti, Nowakowska, Gobessi, Barankiewicz, Salomon-Perzynski, Benvenuti, Efremov, Juszczynski, Lech-Maranda and Muchowicz.This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY).The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.No use, distribution or reproduction is permitted which does not comply with these terms. ", "section_name": "ETHICS STATEMENT", "section_num": null }, { "section_content": "The animal study was reviewed and approved by The Local Ethics Committee for the Animal Experimentation in Warsaw, Warsaw University of Life Sciences, Warsaw, Poland. ", "section_name": "ETHICS STATEMENT", "section_num": null }, { "section_content": "", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.781364/full#supplementary-material Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2022 Goral, Firczuk, Fidyt, Sledz, Simoncello, Siudakowska, Pagano, Moussay, Paggetti, Nowakowska, Gobessi, Barankiewicz, Salomon-Perzynski, Benvenuti, Efremov, Juszczynski, Lech-Maranda and Muchowicz.This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY).The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.No use, distribution or reproduction is permitted which does not comply with these terms. ", "section_name": "SUPPLEMENTARY MATERIAL", "section_num": null } ]
10.18632/oncotarget.25398	Efficient lysis of B-chronic lymphocytic leukemia cells by the plant-derived sesquiterpene alcohol α-bisabolol, a dual proapoptotic and antiautophagic agent	The sesquiterpene α-bisabolol (α-BSB) is a cytotoxic agent against acute leukemia and chronic myeloid leukemia cells. Here the profile of α-BSB citotoxicity was evaluated ex vivo in primary mononuclear blood cells isolated from 45 untreated B-chronic lymphocytic leukemia (B-CLL) patients. We studied the effects of α-BSB by flow cytometric and western blotting techniques with the following findings: (1) α-BSB was an effective proapoptotic agent against B-CLL cells (IC50 42 ± 15 μM). It was also active, but to a lesser extent, on normal residual B cells and monocytes (IC50 68 ± 34 and 74 ± 28 μM, respectively; p < 0.01), while T-cells, though not achieving IC50, were nevertheless decreased. (2) Lipid raft content positively correlated with α-BSB cell sensitivity, while neither the phenotype of B-CLL cells nor the disease clinical stage did affect the sensitivity to α-BSB. (3) Flow cytometry analysis evidenced the induction of pores in mitochondrial and lysosomal membrane after 3- to 5-hour exposure of B-CLL cells to α-BSB, leading to apoptosis; in contrast, western blotting analysis showed inhibition of the autophagic flux. Therefore, according to cellular selectivity, α-BSB is a cytotoxic agent preferentially active against leukemic cells, while its lower activity on normal B cells, monocytes and T cells may account for an additive anti-inflammatory effect targeting the leukemia-associated pro-inflammatory microenvironment. Consistent with the observed effects on intracellular processes, α-BSB should be regarded as a dual agent, both activating mitochondrial-based apoptosis and inhibiting autophagy by disrupting lysosomes.	[ { "section_content": "B chronic lymphocytic leukemia (B-CLL) is the commonest malignancy in the western world.Usually an indolent disease that primarily affects elderly adults, it is characterized by monoclonal CD5 + mature B cells, which accumulate in bone marrow, lymphoid tissues and blood and are mostly blocked in G 0 /G 1 phase of the cell cycle [1,2].Some patients may experience a more aggressive course: secondary hemolytic anemia and/or autoimmune thrombocytopenia may develop, and malignant progression may be seen.Eventually even the most indolent B-CLL leads to bone marrow insufficiency and symptomatic splenomegaly due to neoplastic substitution. Over the last twenty years, a deep revision of its pathogenesis has thoroughly changed the understanding ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "of B-CLL as well as its clinical and therapeutic management [3].The turning-points in this progress can be summarized as follows.(a) Recognition of molecular rearrangements defining more aggressive courses, namely the somatic mutational status [2,[4][5][6][7] and chromosomal alterations such as del11 and 17 [3,8].(b) Understanding of immunoglobulin B-cell receptor [6,9] and microenvironment-driven signals [10][11][12][13][14][15][16][17][18][19][20] pathogenetic potential.(c) Better understanding of kinase-based transduction [10,11,21], programmed cell death and autophagy pathways as well as their gene regulation [14,19,22,23].(d) Availability of new agents (including anti-CD20 and other monoclonal antibodies as well as several kinase inhibitors) that turned out to be more effective than ever before, though perhaps not yet sufficient to cure the disease [24][25][26][27].A better knowledge of the enzymatic pathways regulating adhesion signaling, apoptosis and autophagy as well as related oncogenes has pioneered and fostered the design of and the quest for therapies targeted at components of those pathways.The other way round, the differential efficacy of several of these targeted therapies has shed light on some more relevant mechanisms and transduction pathways in B-CLL [11,25,27].New agents for targeted therapy include specific protein kinase inhibitors, apoptosis activators and autophagy inhibitors, some of them man-projected, some others sorted through existing cellular organic molecules.[3,4,9,19,24].Among these latter, sesquiterpenes [28] like artemisinin [29] gossypol [30] or α-bisabolol (α-BSB) [31] may have practical and theoretical relevance to B-CLL therapy.These multifaceted, virtually limitless, target-restricted therapies should be in compliance with some traditional cornerstones for B-CLL treatment: (a) low toxicity; (b) easily administration preferably orally or subcutaneously; (c) good specificity for the therapeutic targets; (d) opportunity for synergism.In this context, the sesquiterpene alcohol α-BSB is safe at the therapeutic dosages in animal models, can be delivered orally [31], targets specific basic cellular functions like apoptosis and autophagy [22] and is synergistic with some tyrosin kinase inhibitors [32].Here, we investigated the antineoplastic potential of α-BSB in a preclinical model of primary normal and neoplastic cells from untreated B-CLL patients.The great majority were Ig-mutated, normal-caryotype, Binet A-stage males with more than 47 years, more than 12 × 10 -9 /L white blood cells (WBC) and 100 × 10 -9 /L platelets (PLTs), without anemia.We could not establish in this study any different sensitivity to α-BSB related to clinical stage according to Binet or to biological characteristics of B-CLL cells (i.e.CD38 positivity, IgVH mutational state or chromosomal abnormalities). ", "section_name": "Research Paper", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "", "section_name": "Patients", "section_num": null }, { "section_content": "For each patient, we comparatively evaluated the ex vivo sensitivity to α-BSB of B-CLL cells, normal residual B, T lymphocytes and monocytes.2 to 80 μM α-BSB for 24 hours resulted in a dose-dependent reduction of B-CLL cell viability.Figure 1A shows that leukemic lymphocytes IC 50 was 42 ± 15 μM α-BSB, significantly lower than 68 ± 14 and 72 ± 12 μM α-BSB of normal B cells and monocytes, respectively (p = 0.005).Instead, T lymphocytes did not reach the IC 50 in the range of concentrations tested (up to 80 μM α-BSB), and were considered minimally sensitive to α-BSB.Accordingly, Figure 1B shows the preferential depletion of CD5/CD19 B-CLL cells as opposed to the normal mononuclear ones.Thus, α-BSB inhibited leukemic cell viability in a dosedependent manner at concentrations also affecting residual normal B lymphocytes, monocytes and at a lesser extent normal T cells. ", "section_name": "α-BSB inhibits B-CLL cell viability", "section_num": null }, { "section_content": "At the cell membrane level, α-BSB accumulates in the lipid rafts (LRs) [33].Therefore, we sought if the different sensitivity to α-BSB observed in leukemic as compared to normal B lymphocytes could be related to a far more represented LRs in neoplastic cells than in their normal counterparts [34].The largest components of LRs are glycosphingolipids such as ganglioside monosialic 1 (GM1), cholesterol, sphingomielin and phosphatidyl serine.GM1 is a receptor for toxins and viral particles [35,36] whose amounts have been demonstrated to correlate with the response to anti-CD20 therapy in B non-Hodgkin lymphomas and B-CLL [37].Flotillins are another LR component belonging to a family of integral membrane proteins that are actively involved in signal transduction as well as in regulating the motility and localization of LRs [38].Both Flotillin-1 and Flotillin-2 are commonly used as LRs associated markers [39][40][41].In B-CLL cells and in normal B and T lymphocytes of the same sample we quantified cholesterol, GM1 and Flotillin-1 by flow cytometry.Cholesterol was quantified by its affinity to the polyene antibiotic filipin which has fluorescence properties, GM1 by means of its affinity to cholera toxin B (CT-B), and Flotillin-1 by a specific monoclonal antibody [42].As shown in Figure 1C, lipid raft amounts resulted significantly more abundant in CD5/ CD19 B-CLL cells than in normal T and CD19 B cells (p < 0.05 for each comparison).Altogether, these findings correlated with the different sensitivity to α-BSB seen in the different cell subpopulations, accordingly to previous suggestions [33]. ", "section_name": "Lipid raft amounts", "section_num": null }, { "section_content": "To investigate the chain of events leading to the loss of cell viability, we initially stained the B-CLL cells with TO-PRO-3 iodide, which binds with high affinity to double-strand nucleic acids but does not enter intact plasma membrane, and Annexin-V to determine the lipid phosphatidylserine flip from the inner to the outer leaflet of the plasma membrane.We observed a time-dependent increase (data not shown) of TO-PRO-3 and Annexin-V fluorescence when cells were treated with 80 μM α-BSB (Figure 2A).After 3 hours of incubation with α-bisabolol, a substantial proportion of B-CLL cells were Annexin-V-positive, thus signaling the irreversible onset of the apoptotic cascade.Then, to assess the depolarization of plasma membrane upon α-BSB, we used the sensitive anionic dye [Bis-(1,3-Dibutylbarbituric Acid)Trimethin Oxonol] [DiBAC 4 (3)], a slow-response probe that enters the depolarized membrane, with an increase in cell fluorescence.The mean fluorescence intensity (MFI) of DiBAC 4 (3) in B-CLL cells after 1-and 3-hour incubation with 80 μM α-BSB was higher than in control (MFI 1h = 23 ± 7.8 and MFI 3h = 65 ± 13 vs MFI basal = 7.5 ± 0.6; p < 0.001).This indicated the depolarization of plasma membrane, a further hallmark of the apoptotic process induced by α-BSB (Figure 2B).Finally, loading cells with the Ca 2+ indicator Fluo-4 AM, an increased Ca 2+ influx was evident after 20 minutes (Figure 2C).Overall, these results indicated that α-BSB rapidly induced plasma membrane alterations and loss of plasma membrane integrity in B-CLL cells due to apoptosis. ", "section_name": "α-BSB causes damage to plasma membrane", "section_num": null }, { "section_content": "We have previously demonstrated by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetra-ethyl-benz-imidazolyl-carbo-cyanine iodide (JC-1) staining that α-BSB dissipates the ΔΨ m in a variety of cell types [31].Here we investigated the ΔΨ m in α-BSB-treated B-CLL cells.Figure 3A shows that exposure to α-BSB led well-polarized mitochondria to progressively lose their JC-1-dependent red fluorescence, shifting downward, which indicated ΔΨ m dissipation.The calcein-acetoxymethyl ester (calcein-AM) assay explores the mPTP opening, an early event in the damaged cells.Nonfluorescent calcein-AM become fluorescent in cells after cleavage of AM groups via non-specific esterase activity in the cytosol and mitochondria.CoCl 2 cannot enter into healthy mitochondria when the mPTP is closed and it quenches cytoplasmic fluorescence leaving unmodified the mitochondrial one.Figure 3B shows cells that were treated with α-BSB and then loaded with calcein-AM.MFI in treated and untreated cells was 244.5 ± 38 vs 253 ± 17, respectively, but after adding CoCl 2 , MFI was 15.8 ± 5 vs 38.2 ± 3.8, respectively (p < 0.01) due to the quenching of both cytoplasmic and mitochondrial fluorescence in treated cells, as a consequence of the mPTP irreversible opening.Therefore, α-BSB disrupted the membrane permeability of mitochondria, induced ΔΨ m dissipation and triggered the apoptotic death of B-CLL cells. ", "section_name": "α-BSB causes loss of mitochondrial transmembrane potential (ΔΨm)", "section_num": null }, { "section_content": "The effect of α-BSB on lysosomes was studied using the fluorescent lysosomotropic dyes acridine orange (AO) and LysoTracker Green DND-26 (LTG).AO is a metachromatic fluorophore that becomes charged and retained by proton trapping within the lysosomal compartment.When cells are excited by blue light (488 nm blue laser in flow cytometry), lysosomes emit intense red fluorescence while the cytosol and nuclei show weak diffuse green fluorescence.Cells with a reduced number of intact AO-accumulating lysosomes are pale.When lysosomes are damaged, AO is released to the cytosol in monomeric form and it turns green [43,44].The AO-relocation method [44][45][46][47] evaluates the degree of lysosomal damage based on the measure of cytosolic green fluorescence.Instead, the AO-uptake method measures directly the lysosomal red fluorescence.In the α-BSBtreated B-CLL cells, the AO-relocation assay demonstrated an increase in green fluorescence detectable up to 3 hours after treatment (Figure 4A, left), followed by a decrease, likely due to the extracellular release of cytoplasmic AO, secondary to plasma membrane permeabilization.The AOuptake assay demonstrated a decrease in red fluorescence (Figure 4A, right).Notably, this was further confirmed by the LTG-uptake assay aimed to evaluate the lysosomal activity [48,49].Under baseline conditions, LTG was located at large vesicles in the cytoplasm.Treatment induced a time-dependent appearance and increment of a population of dimly fluorescent cells (Figure 4B). ", "section_name": "α-BSB induces pores in lysosomal membrane", "section_num": null }, { "section_content": "Since lysosomes were damaged by α-BSB treatment, we asked whether α-BSB promoted some impairment of the autophagic process, which plays a role in B-CLL cells survival [50].Autophagy is a catabolic process during which cytoplasmic organelles and proteins are nonselectively degraded to rescue cells from death.Double-membranous structures emerge in the cytosol forming an autophagosome that sequesters cytoplasmic proteins and organelles.If the rescue fails, apoptosis is triggered.During autophagy, a cytosolic form of the microtubule-associated protein light chain 3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3phosphatidylethanolamine (LC3-II).This latter is coupled to autophagosomal membranes.The autophagosome then fuses with lysosomes to form autolysosomes, intra-autophagosomal components are broken down by hydrolases and LC3-II is degraded.Hence, LC3-II is an autophagosomal marker for monitoring autophagic activity by immunoblotting or other methods.In evaluating autophagy by LC3-II immunoblotting technique, we observed that α-BSB induced an increment of LC3-II band in B-CLL cells.The increment may indicate either upregulation of autophagosome formation or blockage of autophagic degradation [51][52][53].Figure 4C shows no further accumulation of LC3-II in the presence of pepstatin A and E-64D, inhibitors of the degradation of the autophagic cargo inside the autophagosome.Overall, and similarly to pepstatin or E-64D, α-BSB acts by blocking the autophagic degradation, in keeping with the observed cumulative damage to lysosomal membrane shown in Figure 4A-4B.Therefore, immunoblotting studies of the autophagic process support that α-BSB is an antiautophagic agent that causes the irreversible block of the autophagic flux. ", "section_name": "Autophagic flux inhibition", "section_num": null }, { "section_content": "We and others have previously shown that the natural sesquiterpene alcohol α-BSB induces apoptotic death of a variety of neoplastic cells either in vitro or in animal models [31][32][33][54][55][56][57][58].Notably, we have demonstrated its cytotoxicity against lymphoblastic, myeloblastic and chronic myeloid leukemia cells in preclinical studies [22,31,32].Our present findings show that α-BSB induces efficient lysis of primary B-CLL.The neoplastic CD5/CD19 B cells resulting sensitive to α-BSB were obtained from 45 untreated patients representative of the typical diagnostic characteristics, natural history and clinical outcome of the B-CLL (Table 1).In parallel and in each patient, we analyzed both leukemic cells and normal residual CD5-negative B cells, monocytes and T cells.Our main results may be summarized as follows.a) α-BSB preferentially induced apoptosis of leukemic cells (Figure 1A-1C).b) Concurrently, it had a clear-cut antiautophagic potential (Figure 4).As α-BSB could both promote apoptosis and inhibit autophagy, we may also refer to α-BSB as a dual (proapoptotic, antiautophagic) agent.c) Though at a lesser degree in comparison to leukemic cells, α-BSB had some cytotoxic effect also against normal residual B cells, monocytes and even T cells, which can also account for a plain anti-inflammatory/antimicroenvironment activity (Figure 1A).The differential amounts of lipid raft could possibly help explain the celltype selectivity (Figure 1C). We must place these results in the frame of the natural and therapeutic history of B-CLL.This leukemia has been characterized by a substantial lack of effective treatments until the 1990s.Alkylating agents were the only available therapeutic tools, no more than a symptomatic approach to downsize the neoplastic mass, when needed by symptoms, with no success in inducing disease remission or improving patients' survival [25].Hence, treatment was preferentially delayed until disease-related symptoms occurred, an approach compatible with the usually indolent clinical course of B-CLL [1,24,59].The introduction of purine analogue-based immunochemotherapy incorporating anti-CD20 antibodies was the turning-point that over the last 20 years greatly improved B-CLL treatment in terms of disease remission, failing, however, definitive cure [3,[24][25][26][27].The development of an array of phosphokinase inhibitors was a further, more recent, improvement grounded in a better knowledge of the transduction network downstream BCR, chemokine receptors, receptors for ligands expressed by a variety of microenvironment cells (including monocytes, T lymphocytes, stromal cells etc.), and other receptors on B-CLL cells (including CD19, CD5, CD38 etc.).These agents are administered orally, are often successful even in B-CLL cells bearing unfavorable genetic mutations, and display significant less toxicity than immunochemotherapy [23][24][25][26]. The natural history and the pattern of response to the treatment of B-CLL reflect perhaps the degree of microenvironmental pleiotropic integration of normal mature B lymphocytes [3,13,14,[16][17][18][19][20]60].Though we do not completely understand what processes belong to the basic instruction set of B lymphocytes, it is quite clear that their neoplastic counterpart still respond to strong microenvironmental signals while surviving some death checkpoints, which seem to have lost efficacy: 1) the apoptosis/autophagy balance favors survival, 2) the mitotic checkpoints restraining cell cycle are not efficient and 3) membrane receptors regulating responses to microenviroment-related signals seem to be frozen in an activated configuration that confers a continuous streaming of signaling [20,60]. 1) In the context of apoptosis/autophagy balance, a plausible explanation for the resistance of B-CLL cells to eradication is supported by their autophagic potential, which probably upon treatment allows for the persistency of reservoirs of neoplastic stem cells, in a way somehow similar to that observed for chronic myeloid leukemia CD34 cells [32,61].Therefore, the dual proapoptotic, antiautophagic activity of α-BSB [22] is a point whorthy of interest in medical as well as biological terms, based on the fact that autophagy can rescue from drug-induced death at least some specific type of cancer cells.In particular, a great deal of attention has been paid to the role of autophagy in rescuing cancer stem cells upon treatment, resulting in the therapy-resistance of this neoplastic compartment, which in turn results in the failure of cancer eradication and subsequent possible clinical relapse.It has been shown that the blocking of autophagy allows for the eradication the residual neoplastic disease [61].Moreover, since α-BSB participates to both the induction and inhibition of autophagy while inducing apoptosis [22,31,32], it seems reasonable to situate α-BSB action at the core molecular crossroads regulating both apoptotic and autophagic pathways.The dual action might involve two different layers of control: (a) the α-BSB interaction with the BH3-only protein BID [33], which may deliver signals through modulation of BCL2-Beclin1 interactions at the endoplasmic reticulum leading to Beclin1-dependent autophagy [62] and BID-dependent apoptosis [22,33]; (b) the α-BSB-dependent induction of pores in lysosomal membranes and the collapse of the lysososmal compartment (Figure 4A-4B), which neutralizes the autophagosome function and inhibits the autophagic flux (Figure 4C).Therefore, the core level machinery regulating the crosstalk between autophagy and apoptosis that is targeted and thus highlighted by α-BSB-induced phenomena may lead on the one hand to a better understanding of the role of BCL2-family molecules in oncogenesis and tumor progression, while on the other hand it may represent a target for refined therapeutic tools, such as α-BSB itself and other agents that can affect the apoptosis/autophagy balance [22][23][24].Besides, the possible selective degradation of mitochondria through autophagy, which also occurs, adds further complexity [63].2) If we focus on the cell cycle disregulation, it follows from the above considerations that α-BSB through its cytostatic activity as well as its proapoptotic potential may be expected to overcome the unrestrained cycling of tumor cells in typical indolent forms of B-CLL as well as in more aggressive forms of B-CLL or in more aggressive subclones arising inside the mainly indolent B-CLL cell population.3) If we take the leukemia/microenvironment crosstalk into consideration, our findings suggest that α-BSB may exert pleiotropic activities, inducing both efficient direct B-CLL death and microenvironmental modulation.This latter has at least two facets.First, α-BSB may account for an effect able to hamper the microenvironment-related cell-to-cell signals, especially from T4 cells, supporting B-CLL cell survival [64].Second, the pathogenetic role of the peritumoral inflammatory responses and, therefore, the potential therapeutic significance of targeting them has been variously highlighted over the recent years [11,15,19,20,65,66]; in this context, α-BSB may counteract in an autacoid-like manner the B-, T-and monocyte-dependent pro-inflammatory responses, known to enhance leukemic growth [67].Consequently, the effect of α-BSB on B-CLL is expected to increase over time, ranging from direct cytotoxicity to the maintenance of a microenvironment less favorable to B-CLL growth on the long run, a relevant impact for an agent eligible to be chronically administered.In that respect, α-BSB merits some additional interest on basic clinical grounds such as no or low general toxicity, easy oral delivering and affordable cost.[31,32,68,69]. But at the very heart of our mechanistic views on α-BSB lies a broad biologic issue worthy of consideration.Previous studies have shown that the cytotoxic activity of α-BSB interests a variety of neoplastic cell types [31-33, 54-56, 58], which could indicate an agent targeting basic and quite ancient cellular biochemical pathways, and, according to our findings, the evidence corroborates that α-BSB is an example of xenohormesis involving the function of BH3-only domain BCL2-family proteins at the crossroad of apoptosis and autophagy pathways [22,62,70], though the fine biochemical details of the apoptosis/autophagy crossmachinery targeted by α-BSB remain elusive.Indeed, there is no surprise that these pathways are shared between different types of cancers including leukemias.However, since leukemias, namely acute leukemias, are the most sensitive targets for α-BSB [22,31,32], this likely points to a specific relevance for the evolutionarily conserved apoptosis/autophagy crosstalk machinery in hematopoietic mesodermalderived lineages, and, if we can learn the lesson from the neoplastic counterpart, precisely where leukemia cells arise.We argument that in these cells exclusive unilineage adoption is barred by fusion transcription factors that maintain cells perpetually looping at a low pace without differentiation [64,[71][72][73].As a result their apoptosis/ autophagy machinery is strongly unbalanced towards the autophagic rescue.This allow for the maintenance of an early leukemic compartment highly resistant to eradication, except perhaps for agents targeting autophagy. In conclusion, we provide preclinical evidence that the sesquiterpene α-BSB can promote proapoptotic and antiautophagic activity in B-CLL cells, leading to efficient lysis.Simultaneously, α-BSB has some lytic activity on normal B-lymphocytes, monocytes and T cells, which may entail an overall damping effect against microenvironment components that sustain leukemic cell growth. ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "Viable peripheral blood mononuclear cells (PBMC) of 45 patients with untreated B-CLL were obtained as previously described on a Ficoll-Hypaque gradient from peripheral blood [74,75].The diagnosis of B-CLL was made according to the current guidelines [24] as previously described [10] and fulfilled diagnostic criteria for common B-CLL at the Hematology Section of the Department of Medicine, University of Verona (Verona, Italy), starting from 2002.The study was performed in the context of the project 1828/2010 approved by the ethics committee of the Verona University Hospital and a written informed consent was obtained according to law. ", "section_name": "Patents' cells and ethical requirements", "section_num": null }, { "section_content": "Cells resuspended in RPMI-1640 (Invitrogen, Carlsbad, CA), supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 50 U/mL penicillin and 50 μg/mL streptomycin (complete medium, CM), seeded at a density of 1 × 10 5 cells/mL in 24-well plates and incubated at 37° C in 5% CO 2 were exposed for 24 hours to 20, 40, 80 μM of α-BSB (dissolved in DMSO 1:40; derived from natural organic compounds, purity ≥93%; Sigma-Aldrich, St. Louis, MO), representing the calculated soluble fraction in the assay as reported elsewhere [31].At the end of the culture, the absolute counts of normal and leukemic leukocytes subpopulations were measured with TruCOUNT tubes (Becton Dickinson, San Jose, CA) by flow cytometry according to the manufacturer's instructions with minor modifications.Briefly, 200 μL samples, a mixture of moAbs (CD45-APC-H7, CD5-APC, CD19-PE, CD3-FITC) and 7-amino-actinomycin D for dead cells exclusion (all reagents from Becton Dickinson) were added to the TruCOUNT tubes.After a 15-minute incubation at room temperature, 1 mL lysing reagent (Biosource, Nivelles, BE) was added for 10 minutes.A total of 40,000 beads were acquired on a FACSCanto cytometer (Becton Dickinson) and analyzed by FlowJo 9.3.3software (Tree Star, Ashland, OR) as Dot Plot or PolyChromatic Plot.Hierarchical gating was used to accurately enumerate different populations.Data were expressed as ratio of number of cells treated with α-BSB to number of cells treated with vehicle alone. ", "section_name": "Cytotoxicity assay", "section_num": null }, { "section_content": "Cells were prestained for 10 minutes at room temperature with a mix of moAbs specific for different leukocyte populations (CD3-PerCP, CD5-APC, CD19-APC-H7) and, after washing, the main components of LRs (ganglioside GM1, Flotillin-1 and cholesterol) were quantified.1. GM1.Cells were incubated at 4° C for 10 minutes with Alexa Fluor 488 cholera toxin subunit B (CT-B) (Invitrogen) which specifically binds to the pentasaccharide chain of GM1.Cells were then washed 3 times with PBS and analyzed in flow cytometry.2. Flotillin-1.Cells fixed and permeabilized by a commercial kit (eBiosciences, San Diego, CA) according to the manufacturer's instructions were incubated with an anti-Flotillin-1-Cy3 antibody (Sigma) for 1 hour at 4° C.After washing samples were analyzed in flow cytometry.3. Cholesterol.Cells fixed with component A of Fix&Perm kit (AnDerGrub Bio Research, Kaumberg, AT) were incubated with 100 µg/mL Filipin III (Sigma) for 1 hour at room temperature.After washing samples were analyzed in flow cytometry [37].GM1, Flotillin-1 and cholesterol data were expressed in arbitrary units as the ratio between median fluorescense intensity of stained cells (subtracted of that of unstained ones) and median forward scatter (as a surrogate of cell dimension) multiplied by 10 4 . ", "section_name": "Lipid raft component quantification", "section_num": null }, { "section_content": "", "section_name": "Evaluation of plasma membrane damage", "section_num": null }, { "section_content": "Cells resuspended in CM at 1 × 10 6 /mL were treated with 40 μM α-BSB for 3 and 5 hours at 37° C. 1. Mitochondrial transmembrane potential (ΔΨm).As previously described [31] cells were washed with prewarmed CM, loaded with 4 μM JC-1 (Molecular Probes, Eugene, OR) for 30 minutes then washed twice with PBS.Aliquots of each sample were resuspended in PBS and analyzed by flow cytometry.Visualization of JC-1 monomers and JC-1 aggregates was done using filter sets for fluorescein and rhodamine dyes.Image analysis was done by using Axiovision 3 software.The other aliquot of each sample was resuspended in PBS and analyzed by flow cytometry.2. Mitochondrial permeability transition pore (mPTP).Cells were washed, resuspended in HBSS/ Ca 2+ , loaded with 10 nM calcein-AM with or without 400 μM CoCl 2 for 15 minutes at 37° C (MitoProbe Transition Pore assay kit, Invitrogen) and analyzed by flow cytometry. ", "section_name": "Mitochondrial membrane damage assays", "section_num": null }, { "section_content": "1. Acridine orange-relocation assay.Cells stained with 0,5 μg/mL AO (Molecular Probes) at 37° C for 15 minutes, washed twice, treated with 80 μM α-BSB for 3 hours, and analized by flow cytometry (FL-1 channel).2. AO-uptake assay.Cells incubated with 80 μM α-BSB for 3 hours were stained with 0,5 μg/mL AO for 15 minutes at 37° C, washed twice and analized by flow cytometry (FL-3 channel).3. LysoTracker green-uptake assay.Cells were incubated with 80 μM α-BSB for 1, 3 and 5 hours.Then they were washed and stained at 37° C for 1 hour with 75 nM of the acidotropic dye LTG (Molecular Probes) and analized by flow cytometry (FL-1 channel). ", "section_name": "Lysosome injury assays", "section_num": null }, { "section_content": "5 × 10 6 cells were treated with either DMSO (vehicle) or 80 μM α-BSB for 16 hours.In order to inhibit the degradation of autophagic cargo, Pepstatin A (Sigma, 10 μg/mL) and E-64D (Sigma, 10 μg/mL) were added to the media.Then they were harvested, washed in cold PBS, resuspended in Lysis Buffer (Thermo Scientific, Rockford, IL) plus protease inhibitor cocktail (Sigma).Whole cell lisates were separated on a 14% SDS-PAGE and analyzed by immunoblotting with anti-LC3 antibody and with anti-GAPDH antibody (Cell Signaling Technology, Beverly, MA) and VeriBlot-HRP secondary antibody (Abcam, Cambridge UK).Development was done by enhanced chemiluminescent plus detection reagents (Thermo Scientific) [51]. ", "section_name": "Detection of autophagic flux", "section_num": null }, { "section_content": "Continuous variables were summarized as the mean ± SD.Data were summarized by percentages when appropriate.Student's t-test for means, chi-squared tests, the Mann-Whitney U test, and Kruskall-Wallis analysis of variance (ANOVA) by ranks were used, as appropriate and were considered significant for p values < 0.05.Data were analyzed using the statistical software Stata 12.1 (www.stata.com). ", "section_name": "Statistics", "section_num": null } ]	[ { "section_content": "This article is dedicated to the memory of the biochemist Professor Hisanori Suzuky (1946-2012), a friend worthy of our undying regard and a man, as we may say, the best of all of his time that we have known, and, moreover, the most wise and just (Plato, Phaedo, translated by Henry Cary). ", "section_name": "ACKNOWLEDGMENTS", "section_num": null }, { "section_content": "This work was supported by Italian Association for Cancer Research (AIRC, Milan, Italy)/ Cariverona Foundation (Verona, Italy).FV wants to express its gratitude to Maria Langhieri for supporting cancer research in memory of her mother Iliana Tescaroli. ", "section_name": "FUNDING", "section_num": null }, { "section_content": "B-CLL: B chronic lymphocytic leukemia; α-BSB: α-bisabolol; CM: complete medium; moAbs: monoclonal antibodies; LRs: lipid rafts; GM1: monosialotetrahexosylganglioside; CT-B: cholera toxin B; DiBAC 4 : Bis-(1,3-Dibutylbarbituric Acid) Trimethin Oxonol; MFI: mean fluorescence intensity; JC-1: 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetra-ethyl-benz-imidazolylcarbo-cyanine iodide; ΔΨm: mitochondrial transmembrane potential; mPTP: mitochondrial permeability transition pore; AO: acridine orange; LTG: LysoTracker Green DND-26; LC3-I: microtubule-associated protein light chain 3; LC3-II: LC3-phosphatidylethanolamine; TO-PRO-3: AR performed the research and analyzed results; IF, AB, CT contributed supportive and redational work; AM, CL contributed criticism and suggestions; FV designed the research and wrote the paper.All authors checked the final version of the manuscript. The authors have declared no competing interests exist. ", "section_name": "Abbreviations", "section_num": null }, { "section_content": "B-CLL: B chronic lymphocytic leukemia; α-BSB: α-bisabolol; CM: complete medium; moAbs: monoclonal antibodies; LRs: lipid rafts; GM1: monosialotetrahexosylganglioside; CT-B: cholera toxin B; DiBAC 4 : Bis-(1,3-Dibutylbarbituric Acid) Trimethin Oxonol; MFI: mean fluorescence intensity; JC-1: 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetra-ethyl-benz-imidazolylcarbo-cyanine iodide; ΔΨm: mitochondrial transmembrane potential; mPTP: mitochondrial permeability transition pore; AO: acridine orange; LTG: LysoTracker Green DND-26; LC3-I: microtubule-associated protein light chain 3; LC3-II: LC3-phosphatidylethanolamine; TO-PRO-3: ", "section_name": "Abbreviations", "section_num": null }, { "section_content": "AR performed the research and analyzed results; IF, AB, CT contributed supportive and redational work; AM, CL contributed criticism and suggestions; FV designed the research and wrote the paper.All authors checked the final version of the manuscript. ", "section_name": "Author contributions", "section_num": null }, { "section_content": "The authors have declared no competing interests exist. ", "section_name": "CONFLICTS OF INTEREST", "section_num": null } ]
10.1371/journal.pgen.1009969	Delta/Jagged-mediated Notch signaling induces the differentiation of agr2-positive epidermal mucous cells in zebrafish embryos	<jats:p>Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of <jats:italic>agr2</jats:italic>-positive (<jats:italic>agr2</jats:italic><jats:sup><jats:italic>+</jats:italic></jats:sup>) EMCs in zebrafish embryos during segmentation. We demonstrated that <jats:italic>agr2</jats:italic><jats:sup>+</jats:sup> EMCs contain cytoplasmic secretory granules and express <jats:italic>muc5</jats:italic>.<jats:italic>1</jats:italic> and <jats:italic>muc5</jats:italic>.<jats:italic>2</jats:italic>. Reductions in <jats:italic>agr2</jats:italic><jats:sup>+</jats:sup> EMC number were observed in <jats:italic>mib</jats:italic> mutants and <jats:italic>notch3</jats:italic> MOs-injected <jats:italic>notch1a</jats:italic> mutants, while increases in <jats:italic>agr2</jats:italic><jats:sup>+</jats:sup> cell number were detected in <jats:italic>notch1a-</jats:italic> and <jats:italic>X-Su(H)/ANK</jats:italic>-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of <jats:italic>agr2</jats:italic><jats:sup>+</jats:sup> EMCs. Increased <jats:italic>agr2</jats:italic><jats:sup>+</jats:sup> EMC numbers were also observed in <jats:italic>jag1a</jats:italic>-, <jats:italic>jag1b</jats:italic>-, <jats:italic>jag2a</jats:italic>- and <jats:italic>dlc</jats:italic>-overexpressing, but not <jats:italic>jag2b</jats:italic>-overexpressing embryos. Meanwhile, reductions in <jats:italic>agr2</jats:italic><jats:sup>+</jats:sup> EMC numbers were detected in <jats:italic>jag1a</jats:italic> morphants, <jats:italic>jag1b</jats:italic> mutants, <jats:italic>jag2a</jats:italic> mutants and <jats:italic>dlc</jats:italic> morphants, but not <jats:italic>jag2b</jats:italic> mutants. Reduced numbers of <jats:italic>pvalb8</jats:italic>-positive epidermal cells were also observed in <jats:italic>mib</jats:italic> or <jats:italic>jag2a</jats:italic> mutants and <jats:italic>jag1a</jats:italic> or <jats:italic>jag1b</jats:italic> morphants, while increased <jats:italic>pvalb8</jats:italic>-positive epidermal cell numbers were detected in <jats:italic>notch1a-</jats:italic>overexpressing, but not <jats:italic>dlc</jats:italic>-overexpressing embryos. BrdU labeling further revealed that the <jats:italic>agr2</jats:italic><jats:sup>+</jats:sup> EMC population is maintained by proliferation. Cell lineage experiments showed that <jats:italic>agr2</jats:italic><jats:sup><jats:italic>+</jats:italic></jats:sup> EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of <jats:italic>agr2</jats:italic><jats:sup>+</jats:sup> EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.</jats:p>	[ { "section_content": "Mammalian skin is composed of four stratified epithelial layers, including the stratum basale, stratum spinosum, stratum granulosum and the outermost stratum corneum [1].Notably, the stratum corneum consists of layers of dead corneocytes that serve as an inert hydrophobic boundary between the animal and the environment.Together the stratified epithelia of skin epidermis form a complex barrier that protects terrestrial vertebrates from dehydration, pathogen infection and mechanical stress.Several transcription factors and signaling pathways (p63, KLF4, IRF6, Grhl3, MAPK cascade and Notch signaling) have been implicated in epidermal stratification, renewal and differentiation [2][3][4][5][6][7][8]. In contrast to mammalian species, which are mostly terrestrial, teleosts live in aquatic habitats, where they encounter ionic, osmotic and acid-base fluctuations as well as infectious pathogens.To cope with these environmental challenges, teleost skin is composed of living cells covered with a layer of mucus [9].In zebrafish, the embryonic epidermis consists of a surface enveloping layer, which develops into periderm, and an inner epidermal basal layer containing both undifferentiated and differentiated cells (ionocytes and mucous cells) [10].In the adult zebrafish, the epidermis consists of three layers: a surface layer of keratinocytes, an intermediate layer composed of ionocytes, mucous cells, club cells and undifferentiated cells, and a basal layer of undifferentiated cells. In the skin of embryos and the gills of adult fish, specialized ionocytes serve to maintain body fluid ionic homeostasis [11,12].Both ionocytes and keratinocytes are derived from common precursors, which are located in the ventral non-neural ectoderm and express a dominant-negative form of p63 (4Np63) at the late gastrula stage of zebrafish embryos [13].Whether cells from p63 + ventral ectoderm become ionocytes or keratinocytes is determined by Delta-Notch lateral inhibition [14,15].Epidermal cells that express high levels of Dlc ligand develop into ionocyte progenitors.The Dlc ligand interacts with Notch1a/3 receptors on adjacent epidermal cells to prevent dlc expression in those cells, causing them to become keratinocytes.Notably, the ionocyte progenitor population was recently shown to be maintained by Klf4 via its regulation of p63 + epidermal stem cell proliferation and DeltaC-Notch lateral inhibition [16].The development of different types of ionocytes, including Na + , K + -ATPase-rich cells (NaRCs) and H + -ATPase-rich cells (HRCs), is further mediated by the foxi3a/foxi3b regulatory loop [14]. A third type of epidermal cells is mucous cells.The major components of mucus are mucous cell-secreted Mucin glycoproteins, which form a network of polymers joined by disulfide bridges [17].Innate immune-related proteins (antimicrobial peptide and apolipoprotein A-I) are also found in fish epidermal mucus [18][19][20].Thus, mucus is considered to be the first line of defense against pathogen invasion; however, it remains unclear how epidermal mucous cells (EMCs) develop in fish.A recent study showed that pvalb8 (a putative EMC marker), atp1b1b (a NaRC marker) and atp6v1aa (a HRC marker, previously named atp6v1al) display complementary punctate expression patterns in zebrafish embryos at 24 hours post-fertilization (hpf) [21].Furthermore, knockdown of foxi3a caused loss of atp1b1b + ionocytes and expansion of pvalb8-positive cells, suggesting that pvalb8-positive cells and ionocytes are derived from common non-keratinocyte precursors [21].Previously, we found that the gene, anterior gradient 2 (agr2), is expressed in most zebrafish organs that contain mucus-secreting cells, including the intestine and epidermis of embryos [22].Importantly, knockdown of agr2 was shown to disrupt terminal differentiation of intestinal goblet cells in zebrafish embryos [23], and maturation of intestinal goblet cells is known to be regulated by Foxa2 and Hif1abinduced agr2 expression [24].We were therefore interested to determine whether agr2 + EMCs are derived from non-keratinocyte precursors, like pvalb8-positive cells. In this study, we first demonstrated that agr2 + EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2.Then, we found that the numbers of agr2 + EMCs were reduced in mib mutants and notch3 MOs-injected notch1a mutants.On the other hand, the agr2 + EMC numbers were increased in notch1a-and X-Su(H)/ANK-overexpressing embryos.Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2 + EMCs.Next, we determined that agr2 + EMC numbers were increased in jag1a-, jag1b-, jag2a-and dlc-overexpressing, but not jag2b-overexpressing embryos, while agr2 + EMC numbers were diminished in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants.Reductions in pvalb8-positive epidermal cell numbers were also observed in mib mutants, jag1a morphants, jag1b morphants and jag2a mutants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos.BrdU labeling was then used to show that the agr2 + EMC population is maintained by proliferation.Cell lineage experiments further demonstrated that agr2 + EMCs and keratinocytes or ionocytes are derived from common ectodermal precursors. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Initially, we conducted in situ hybridization to find the earliest stage at which agr2 + EMCs emerge.At 15 s, agr2 began to be expressed in the epidermis of the developing yolk extension, in addition to its expression in the otic vesicles, hatching gland and future anus (Fig 1Aa).Increased signal for agr2 + EMCs was then detected in both tail and yolk extension during 18 s and 19 hpf stages (Fig 1Ab and1Ac).Agr2 + EMCs were detected in the yolk sac from 20 hpf and in the trunk, tail and different yolk regions at 22 hpf (Fig 1Ad -1Af). Previously, we investigated the mechanisms controlling agr2 expression in the pharynx and intestine of zebrafish; in doing so, we generated a Tg(-6.0 k agr2:EGFP) transgenic line, which recapitulated endogenous agr2 expression [24].In order to confirm whether EGFP in the reporter line colocalizes with Agr2 in EMCs, we performed double immunofluorescence staining using anti-salmon Agr2 and anti-GFP antibodies.Colocalization of Agr2 and EGFP was detected in the EMCs within the yolks and trunks of Tg(-6.0 k agr2:EGFP) transgenic embryos at 48 hpf (Fig 1B). In previous studies, pvalb8-positive cells were identified as putative EMCs in zebrafish embryos [21,25].We therefore wondered whether agr2 and pvalb8 are co-expressed in the same epidermal cells.To answer this question, we conducted double fluorescence in situ hybridization on Tg(-6.0 k agr2:EGFP) transgenic embryos.Distinct epidermal cells expressing egfp/agr2 or pvalb8 were identified in the trunk and yolk sac at 24 hpf, indicating that agr2 + EMCs and pvalb8-positive cells may represent two different populations of EMCs (S1 Fig). Since intestinal goblet cells mostly secrete Muc2 glycoprotein to create a mucus layer [26], we investigated which mucin genes are expressed in the EMCs.We found that muc5.1/5.2 were co-expressed with egfp in the EMCs within the trunks and yolks of Tg(-6.0 k agr2:EGFP) embryos at 48 hpf (Fig 1C). To examine the ultrastructure of EMCs, we conducted transmission electron microscopy analyses and immunogold labeling on 72 hpf Tg(-6.0 k agr2:EGFP) embryos (Fig 1D).The cytoplasm of each EMC was filled with secretory granules surrounded by endoplasmic reticulum, and the nucleus was located at one side of the cell (Fig 1Da).GFP immunogold electron microscopy staining showed that gold-labeled GFP was distributed in the rough endoplasmic reticulum and lumen, cytoplasm and secretory granules (Fig 1Db and1Dc).Together, these results indicated that agr2 + EMCs develop during mid-segmentation stages and secrete Muc5.1/5.2 glycoprotein to form a mucus layer.Moreover, agr2 + EMCs and pvalb8-positive cells may represent two different populations of EMCs. ", "section_name": "Development of agr2-expressing EMCs", "section_num": null }, { "section_content": "Since Delta-Notch-mediated lateral inhibition determines the cell fates of ionocytes and keratinocytes [14,15], we wondered whether Notch signaling is also involved in the development of agr2 + EMCs.We conducted in situ hybridization using agr2 RNA probes and observed a significant reduction in agr2 + EMC numbers in the yolks and trunks of mib ta52b and mib tfi91 mutants compared to respective sibling wild-type embryos at 24 hpf (Fig 2Aa -2Af).Of note, the reduction of agr2 + EMC number was higher for mib ta52b mutants (95.6%) than that for mib tfi91 mutants (76.6%), which is consistent with the fact that mib ta52b is an antimorphic allele [27].Additionally, we observed a substantial decrease in pvalb8-positive cell number in the yolks and trunks of mib ta52b and mib tfi91 mutants compared to respective sibling wild-type embryos at 24 hpf, although reduction levels were less than those of agr2 + EMC number (S2A Fig). We next investigated the timing at which agr2 + EMCs require Notch signaling.We incubated zebrafish embryos with a γ-secretase inhibitor (MK0752) at different developmental stages to evaluate the effects on agr2 + EMCs.Initially, we treated embryos with 100 μM MK0752 for the intervals of bud (10 hpf)-17 hpf, 10-20 hpf or 10-24 hpf.We observed substantial reductions in agr2 + EMC numbers in the trunks and yolks compared to embryos treated with DMSO from 10-24 hpf (S2B Fig) .In order to find the required minimal treatment time, we treated embryos with 50 μM MK0752 from 10-13 and 10-14 hpf or 11-14 and 11-15 hpf.We found that 3 h of MK0752 treatment did not affect agr2 + EMC number in the trunks and yolks, whereas 4 h of MK0752 treatment significantly reduced agr2 + EMC numbers in these regions compared to DMSO-treated embryos (S2C Fig) .We then treated embryos with 75 μM MK0752 from 10-15, 10-16 and 10-17 hpf and observed incremental reductions of agr2 + EMC numbers in the trunks and yolks compared to embryos treated with DMSO from 10-17 hpf (Fig 2B).Substantial decreases were seen in total agr2 + EMC number and EMC number in the yolk (but not in the trunk) in 12-17 hpf-treated embryos; however, these reductions were less than those detected in MK0752-treated embryos from 10-15 hpf.Together, these results indicate that Notch signaling is essential for the development of agr2 + EMCs and pvalb8-positive cells.Furthermore, Notch signaling is required specifically from 10 to 15 hpf for the development of agr2 + EMCs.Jagged-mediated activation of Notch signaling is required for the differentiation of agr2 + EMCs Since Notch1a and Notch3 are determinants of cell fate for ionocytes and keratinocytes [14], we evaluated whether overexpression of notch1a, notch3 or a dominant activator of Notch signaling, X-Su(H)/ANK [28], affects the development of agr2 + EMCs.The agr2 + EMC numbers were elevated in the trunks and yolks of embryos injected with 200 pg notch1a intracellular domain (ICD) but not LacZ mRNA at 19 and 30 hpf (Figs 3Aa-3Ac and S3Aa-S3Ac).Similarly, significantly increased agr2 + EMC numbers were observed in the trunks and yolks of embryos injected with 10 pg X-Su(H)/ANK mRNA compared to LacZ-injected controls at 24 hpf (Fig 3Ad -3Af).Also, the pvalb8-positive cell numbers were increased in the trunks and yolks of embryos injected with 200 pg notch1a ICD but not LacZ mRNA at 24 hpf (S3Ad-S3Af Fig) .These data suggested that activation of Notch signaling via overexpression of notch1a ICD or X-Su(H)/ANK results in the increased trunk and yolk agr2 + EMC numbers.In addition, Notch signaling activation by notch1a ICD overexpression promoted increase of pvalb8-positive cell numbers in the trunks and yolks. In contrast, agr2 + EMC number was diminished in embryos injected with various doses (10 to 200 pg) of notch3 ICD compared to LacZ-injected controls at 24 hpf (S3B Fig) .Interestingly, this result is similar to a previous in vitro finding that mouse Notch3 ICD inhibits Notch1 ICD-induced HES expression in a concentration dependent manner [29].Thus, overexpressed zebrafish Notch3 ICD may also function as a repressor of endogenous Notch1a ICD to reduce the number of agr2 + EMCs. We next investigated whether deficiency of notch1a or notch3 affects the development of agr2 + EMCs.The numbers of agr2 + EMCs in the trunks and yolks were similar in notch1a th35b homozygous mutants and sibling wild-type embryos at 24 hpf (Fig 3Ba , 3Bb, and3Bg).Likewise, comparable trunk and yolk agr2 + EMC numbers were observed in 0.8 ng notch3 UTRMO-or 2 ng notch3 SPMO-injected and wild-type embryos at 24 hpf (Figs 3Bj-3Bl and S3C).We next investigated whether the lack of both notch1a and notch3 affects the development of agr2 + EMCs.The agr2 + EMC numbers in the trunks and yolks of notch1a th35b homozygous mutants injected with suboptimal doses (0.4 ng and 0.6 ng) of notch3 UTRMO were greatly reduced compared to sibling controls injected with the same amounts of MO (Fig 3Bc-3Bf, 3Bh, and 3Bi).Similar reduction of agr2 + EMC number was observed in the trunks and yolks of notch1a th35b homozygous mutants injected with suboptimal doses (1 ng and 1.5 ng) of notch3 SPMO (S3D Fig) .These results indicate that both Notch1a and Notch3 are required for the differentiation of agr2 + EMCs. Zebrafish has four Jagged ligands, and jag2a and jag2b are expressed in the epidermis of yolk sac in a punctate pattern at 11 s stage [15], so we next investigated which Jagged ligand is involved in the development of agr2 + EMCs by modulating their expression in zebrafish embryos.Injection of 300 pg jag1b but not LacZ mRNA substantially increased agr2 + EMC numbers in the trunks and yolks of embryos at 24 hpf (Fig 4Ad -4Af).While injections of 250 pg jag1a or 200 pg jag2a did not increase agr2 + EMC numbers in the trunk, the agr2 + EMC numbers in the yolk and total EMCs were elevated when compared to LacZ-injected embryos at 24 hpf (Fig 4Aa -4Ac, and4Ag-4Ai). We then investigated whether deficiency of different Jagged ligands affects the development of agr2 + EMCs.Reduced numbers of trunk and yolk agr2 + EMCs were observed in jag1b jj59 and jag2a sa10076 homozygous mutants (Fig 4Be -4Bg and4Bi-4Bk) as well as in jag1b and jag2a morphants (Fig 4Bh and4Bl) compared to wild-type embryos at 24 hpf.Substantially decreased agr2 + EMC numbers in the trunks and yolks were also observed in embryos injected with jag1a UTR MO or jag1a SPMO, compared to wild-type embryos (Fig 4Ba -4Bd).In contrast, trunk and yolk agr2 + EMC numbers were similar in jag1a sa168 homozygous mutants and sibling wild-type embryos (S4A Fig) .We also evaluated whether the jag1a sa168 mutant is a null allele.We performed DNA sequencing to confirm that jag1a sa168 homozygous mutants harbor a G to T mutation in the gene, which is predicted to encode a truncated Jag1a protein with MNNL, DSL and six EGF domains.Expression levels of jag1a, jag1b, jag2a or jag2b were not affected in jag1a sa168 homozygous mutants according to RT-qPCR (S4B Fig) .We then knocked down jag1b and jag2a in wild types and jag1a sa168 mutants.Similar reductions in the numbers of trunk and yolk agr2 + EMCs were identified in jag1b and jag2a MOs-injected wildtype and jag1a sa168 mutants, compared to un-injected controls at 24 hpf (S4C Fig) .Therefore, we suspect that mutated Jag1a protein in jag1a sa168 homozygous mutants still functions normally, at least in terms of agr2 + EMC development.Similarly, no alterations of agr2 + EMC numbers in the trunks and yolks were detected after injection of 500 pg jag2b or in jag2b sa1720 heterozygous or homozygous mutants as compared to LacZ-injected or sibling wild-type embryos at 24 hpf (S4D Fig). Low but significant decreases in total agr2 + EMC numbers were detected in jag1b jj59 (19.9%) and jag2a sa10076 (14.6%) homozygous mutant embryos, suggesting that these Jagged ligands have redundant functions.We investigated whether knockdown of jag1a, jag2a or both genes in jag1b jj59 homozygous mutants could further reduce agr2 + EMC numbers.Compared to un-injected jag1b jj59 homozygous mutants, a 24.8% decrease in the total agr2 + EMC number was detected in jag1b jj59 homozygous mutants injected with both jag1a and jag2a MOs at 24 hpf (Fig 4C).This result suggests that jag1a, jag1b and jag2a function redundantly during the development of agr2 + EMCs.We also evaluated whether deficiency of jag1a, jag1b or jag2a affects the development of pvalb8-positive cells.Similarly, low but significant reductions in trunk and yolk pvalb8-positive cell numbers were detected in jag1a morphants, jag1b morphants and jag2a mutants (S4E Fig). Together, these results demonstrate that Jag1a/1b/2a-mediated activation of Notch1a/3 signaling induces the differentiation of agr2 + EMCs.Likewise, Jagged1a/1b/2a-mediated activation of Notch1a signaling also results in the differentiation of pvalb8-positive cells. ", "section_name": "Proper Notch signaling is required for the differentiation of agr2 + EMCs", "section_num": null }, { "section_content": "Previous studies showed that keratinocytes and ionocytes are derived from common precursors from ventral ectoderm [15].To investigate whether agr2 + EMCs, kerationocyte and ionocytes have the same embryonic origin, we conducted cell-lineage experiments.We transplanted single ventral cells from embryos that had been injected with fluorescent dextran at the 1-cell stage homotypically into unlabeled recipient embryos at shield stage.Chimeric embryos were fixed at 24 hpf and stained with anti-p63 antibody (labels keratinocytes) and anti-salmon Agr2 antibody, or the embryos were stained with anti-Na + -K + -ATPase antibody and anti-salmon Agr2 antibody (Fig 5A).We identified one embryo (1/7) in which the clones of labeled descendants comprised three p63-positive cells, four Agr2-positive cells and three Agr2/P63-negative cells (Fig 5Aa -5Ae).These results indicate that agr2 + EMCs and keratinocytes are indeed derived from common ectodermal precursors.Similarly, we identified one embryo (1/10) in which the clones of labeled cells consisted of two Agr2-positive cells, three Na + -K + -ATPase-positive cells, and three Agr2/Na + -K + -ATPase-negative cells (Fig 5Af -5Ai).These results demonstrate that agr2 + EMCs and ionocytes are also derived from common ectodermal precursors. Since jag1a, jag1b and jag2a are required for the development of agr2 + EMCs (Fig 4) and jag2b was shown to be co-expressed with foxi3a and foxi3b in ionocyte progenitors at 11 s stage [15], we further conducted double in situ hybridization for foxi3a or grhl1 with jag1a, jag1b or jag2a, as well as immunofluorescence staining of p63 with fluorescence in situ hybridization for jag1a, jag1b or jag2a at 5 s stage (Fig 5B).jag1a exhibited weak expression in a punctate pattern in the epidermis at 5 s (Fig 5Bb), while jag1b and jag2a showed prominent puncatate expression in the epidermis at 5 s and 10 s (Figs 5Bg and S5Cb, S5Cg, S5Ch).Consistent with previous findings, co-expression of foxi3a with jag1a, jag1b or jag2a was identified in the majority of ionocyte progenitors at 5 s.Many jag1a + /foxi3a -cells, jag1b + /foxi3a -cells or jag2a + /foxi3a -cells were also identified at this stage (Fig 5Bc -5Be).Similarly, co-expression of grhl1 with jag1b, jag1a or jag2a was identified in the majority of non-keratinocyte epidermal cells at 5 s.Many jag1b + /grhl1 -cells, jag1a + /grhl1 -cells or jag2a + /grhl1 -cells were also identified at this stage (Fig 5Bh -5Bj).A previous study showed that foxi3a is expressed in a sub-population of p63 + epidermal stem cells at bud stage [14], and we also found that jag1a, jag1b and jag2a are expressed in some p63 + epidermal stem cells at 5 s stages (Fig 5Bm -5Bo). We next compared the expression sites of agr2 + EMCs with foxi3a, three jagged ligands or grhl1 by double in situ hybridization (Fig 5C).Although some foxi3a + ionocytes were localized adjacent to agr2 + EMCs in the yolks and trunks at 24 hpf (Fig 5Cc and5Cf), we suspect this close proximity may be a random event because knockdown of foxi3a did not affect the development of agr2 + EMCs (see S5A Fig) .Additionally, we found that approximately 87.5±1.8%jag1a + (n = 5), 86.0±3.0%jag1b + (n = 4) and 88.4±4.6% jag2a + (n = 6) epidermal cells were localized next to agr2 + EMCs at 22 hpf (total yolk agr2 + EMC number was used as the denominator; Fig 5Cg -5Cl), suggesting that agr2 + EMCs may receive Notch signals from neighboring Jagged-expressing cells.Interestingly, co-expression of agr2 with grhl1 was observed in the majority of agr2 + EMCs at 22 hpf (Fig 5Cm -5Co); this indicates that grhl1 is a common marker for all non-keratinocyte epidermal cells including newly-identified agr2 + EMCs and the previously-identified pvalb8-positive cells and foxi3a + /foxi3b + ionocyte progenitors [21]. Co-expression of foxi3a and jag1b, jag1a or jag2a was observed in ionocyte progenitors at 5 s (Fig 5Ba -5Be), suggesting a potential role of ionocytes in the development of agr2 + EMCs.We then investigated whether trunk and yolk agr2 + EMC numbers were affected in foxi3a morphants at 24 hpf (S5A Fig) .Loss of atp6v1aa-positive ionocytes was identified in foxi3a morphants compared with wild-types and control embryos injected with foxi3a misMO (S5Ae-S5Ag Fig) .Unexpectedly, comparable numbers of agr2 + EMCs in the trunk and yolk were observed between foxi3a morphants and wild-types.However, low level but significant increases in trunk and total agr2 + EMC numbers were identified in foxi3a morphants compared with foxi3a misMO-injected control embryos, which may be due to a toxic effect of the foxi3a misMO injection (S5Aa-S5Ad Fig). Since co-expression of grhl1 and jag1b, jag1a or jag2a was also observed at 5 s (Fig 5Cm-5Co), we further investigated whether knockdown of grhl1 affected trunk and yolk agr2 + EMC numbers at 24 hpf (S5B Fig) .Similar numbers of agr2 + EMCs in the trunks and yolks were observed among wild-type embryos and those that had been injected with grhl1 ATGMO or grhl1 SPMO (S5Ba-d Fig) .Consistent with a previous study showing that Grhl1 negatively regulates its own transcription [21], embryos injected with grhl1 ATGMO or grhl1 SPMO displayed higher numbers of grhl1-positive cells than wild-type controls (S5Be-S5Bg Fig) .These results indicate that ionocytes and grhl1-positive cells are not the origin of the Jagged signals that promote the development of agr2 + EMCs. Since Dlc-Notch-mediated lateral inhibition is essential to specify epidermal ionocyte progenitors from epidermal stem cells [14], we wondered whether Dlc participates in the specification of agr2 + EMCs.Our previous study showed that dlc + ionocyte progenitor numbers reached a maximum at bud stage and were decreased at the 5 s stage [16], so we next conducted double in situ hybridization for dlc with jag1b, jag1a or jag2a at 5 s and 10 s stages Co-expression of dlc with jag2a, jag1a or jag1b was observed in some ionocyte progenitors at 5 s and 10 s (S5Ca-S5Ce and S5Cj Fig) .Nevertheless, many jag1b + /dlc -cells, jag1a + /dlc -cells, or jag2a + /dlc -cells were identified in the yolk at these two stages (S5Cc-S5Ce, S5Ci, and S5Cj Fig) .We then investigated whether deficiency of dlc affects trunk and yolk agr2 + EMC numbers (S5D Fig) .Comparable numbers of agr2 + EMCs in the trunks and yolks were identified in dlc tm98 mutants and sibling wild-type embryos at 24 hpf (S5Da-S5Dc Fig) .Since dlc tm98 mutant is a hypomorphic allele [30], we then investigated the effects of dlc morpholino knockown on trunk and yolk agr2 + EMC numbers [31].Unexpectedly, significant reductions in the trunk and total agr2 + EMC numbers were detected in embryos injected with dlc MO compared to wild-type embryos at 24 hpf (S5Dd-f Fig) .As in the previous experiments, increases in foxi3a + ionocyte numbers were observed in dlc morphants compared with wild-types at bud stage (S5Dg and S5Dh Fig) .We next investigated whether overexpression of dlc mRNA affects the development of agr2 + EMCs.Increased numbers of trunk or yolk and total agr2 + EMCs were detected in embryos injected with 92 or 184 pg dlc mRNA but not LacZ mRNA at 24 hpf (S5Di-S5Dl Fig) .However, similar trunk and yolk pvalb8-positive cell numbers were observed in embryos injected with 92 or 184 pg dlc mRNA or LacZ mRNA at 24 hpf (S5Dm-S5Do Fig) .We also investigated whether quadruple MO-mediated knockdown of dlc, jag1a, jag1b and jag2a could further reduce trunk and yolk agr2 + EMC numbers.Compared to wild-type embryos, a 23.8% decrease in the total agr2 + EMC number was detected in embryos injected with dlc, jag1a, jag1b and jag2a MOs (S5Dp-S5Dr Fig) .These results suggest that Dlc also participates in the differentiation of agr2 + EMCs but not pvalb8-positive cells. Since agr2 + EMC numbers increased with embryonic growth, we wondered how the population is maintained after differentiation.We conducted BrdU labeling and agr2 in situ hybridization or Agr2 immunofluorescence on wild-type embryos at 26 s and 24 hpf or Together, these results suggest that some p63 + epidermal stem cells, but not ionocytes or grhl1-positive cells, are the Jagged-expressing cells that activate Notch1a/3 signaling to induce the development of agr2 + EMCs.Then, after differentiation, agr2 + EMCs proliferate to maintain the population. ", "section_name": "agr2 + EMCs, ionocytes and keratinocytes are derived from a common nonneural ectodermal precursor", "section_num": null }, { "section_content": "Among the differentiated epidermal cells in teleosts (keratinocytes, ionocytes and mucous cells), ionocytes and EMCs are evenly dispersed throughout the tissue.The specification of ionocytes begins at late gastrulation stage via Dlc-Notch1a/3-mediated lateral inhibition [14].In this report, we demonstrate that Dlc/Jag1a/1b/2a-mediated activation of Notch1a/3 signaling directly or indirectly induces the differentiation of EMCs expressing agr2 during mid-segmentation stages.We also show that the agr2 + EMC population is maintained by proliferation (Fig 6). We showed that agr2 + EMCs appear initially in the yolk extension at 15 s and in the tail, trunk and yolk sac from 15 s to 22 hpf.Similar to intestinal goblet cells, agr2 + EMCs contain secretory granules and synthesize Muc5.1/5.2 glycoprotein to form a mucus layer (Fig 1).Dramatically reduced numbers of agr2 + EMCs in the yolks and trunks were identified in two mib mutants defective in Notch signaling.Furthermore, treatment with a γ-secretase inhibitor also indicated that differentiation of agr2 + EMCs requires Notch signaling specifically from 10 to Dlc and Jag1a/1b/2a are two alternate ligands of Notch signaling and they specify two major cell types in the epidermis via a streamlined Notch-dependent process.The specification of ionocytes by Dlc-Notch1a/3 mediated lateral inhibition occurs early, during late gastrulation stage [14], while Jag1a/1b/2a-mediated Notch1a/3 activation induces differentiation of agr2 + EMCs later, during mid-segmentation stages (Fig 6B).Although Dlc is mainly responsible for the first specification event, knockdown of dlc decreased trunk and total agr2 + EMC numbers, and overexpression of dlc increased trunk or yolk and total agr2 + EMC numbers (S5D Fig) .These results indicate that Dlc also participates in the differentiation of agr2 + EMCs.Interestingly, a 23.8% decrease in the total agr2 + EMC number was detected in embryos injected with four MOs against dlc, jag1a, jag1b and jag2a; this level of decrease is similar to that (24.8%) observed in jag1b jj59 homozygous mutants injected with both jag1a and jag2a MOs (Figs S5D and4C).This result may be due to that dlc + ionocyte progenitor cell numbers reached a maximum at bud stage and were diminished at 10 s stage (S5C Fig) [16].In contrast, epidermal cells expressing jag1a, jag1b or jag2a were identified in embryos at 5 s ( [32].Although co-expression of dlc or foxi3a with jag1a, jag1b or jag2a was observed at 5 s stage (Figs 5B and S5C), the identified cells may not participate in the specification of ionocytes because no alterations in numbers of atp1b1b + or atp6-v1aa + ionocytes were observed in embryos injected with single or quadruple MOs against all four jag genes [15].Expression of various jag genes occurs during 5 s to 10 s stage, which is also in line with the conclusion that Notch signaling is required from 10 to 15 hpf for the development of agr2 + EMCs (Fig 2).In addition, epidermal cells expressing jag1a, jag1b or jag2a were localized nearby agr2 + EMCs at 22 hpf (Fig 5C).These results support the conclusion that the second specification event is initiated by Jag1a/1b/2a ligands. Although coexpression of three jag ligands was identified in ionocyte progenitor and nonkeratinocyte markers, knockdowns of foxi3a or grhl1 do not affect the development of agr2 + EMCs (S5A and S5B Fig) .Nevertheless, we found coexpression of jag1a, jag1b and jag2a in some p63 + epidermal stem cells at 5 s (Fig 5B ), suggesting that p63 + epidermal stem cells are the Jag1a, Jag1b, and Jag2a presenting cells for agr2 + EMC differentiation.A previous publication reported that expression of human JAG1 and JAG2 was upregulated by p63 and a p63-binding site in the JAG1 gene that interacts with p63 protein was identified in vivo [33].These findings further support the idea that p63 + epidermal progenitor cells expressing Jag1a, Jag1b, and Jag2a are responsible for inducing the formation of agr2 + EMCs in neighboring cells.While our model indicates sequential specification of ionocytes/kerationocytes and agr2 + EMCs via Dlc-Notch1a/3-mediated lateral inhibition and Dlc/Jag1a/1b/2a-mediated Notch 1a/3 activation (Fig 6B ), a variant of this model could also be proposed.That is, early developed p63 + keratinocytes may begin to express Jag1a/1b/2a during bud to 15 hpf and supply Notch1a/3 signal that activates differentiation of neighboring epidermal cells into agr2 + EMCs.However, the cell fates of these jag1a/1b/2a-expressing epidermal cells are still which sequential specification of ionocytes/keratinocytes and agr2 + EMCs/pvalb8-positive cells occurring in zebrafish embryos.The previously-explored specification of ionocytes and keratinocytes via Dlc-mediated Notch1a/3 lateral inhibition occurs at late gastrulation stage, while the newly-identified induction of agr2 + EMCs by Dlc and Jag1a/1b/ 2a-mediated activation of Notch1a/3 signaling and the induction of pvalb8-positive cells via Jag1a/Jag1b/Jag2amediated activation of Notch1a signaling occur during segmentation stage.Underlying data are available in S1 Data. https://doi.org/10.1371/journal.pgen.1009969.g006unknown (Fig 6B).Single cell RNA seq of 4Np63-lineage traced epidermal cells may provide definitive answers in the future. Additionally, we showed that agr2 + EMCs and pvalb8-positive cells may represent two different populations of EMCs (S1 Fig) .Significantly reduced trunk and yolk pvalb8-positive cell numbers were observed in mib ta52b and mib tfi91 mutants as well as jag1a morphants, jag1b morphants and jag2a mutants (S2A and S4E Figs), while increased trunk and yolk pvalb8-positive cell numbers were identified in notch1a-overexpressing, but not dlc-overexpressing embryos (S3A and S5D Figs).Unlike pvalb8-positive cell numbers, which were negatively regulated by Foxi3a [21], the agr2 + EMC numbers in foxi3a morphants and wild-type embryos were similar at 24 hpf (S5A Fig) .Such differential regulation of Foxi3a on numbers of pvalb8positive cells and agr2 + EMCs remains to be studied in the future.Nevertheless, our results suggest that Jag1a/1b/2a-mediated Notch signaling activation might participate in the development of mucous cell types other than agr2 + EMCs, such as pvalb8-expressing putative EMCs. Although several major features of teleost and mammalian epithelia are different, both function to protect organisms from environmental challenges.Moreover, the activation of Notch1 signaling by Delta-1 or Jagged-1 has been implicated in early and late events of keratinocyte differentiation within mammalian epidermis [34,35].Within the stratified epithelium, Notch signaling induces terminal differentiation of spinous cells in a Hes1-independent manner; Notch signaling also promotes Ascl2 transcription to induce granular differentiation, which is repressed by Hes1 protein [36].Similarly, we showed that activation of Notch1a/3 signaling by Dlc/Jag1a/1b/2a promote the differentiation of agr2 + EMCs in zebrafish embryos.Therefore, Notch signaling appears to exert an essential and conserved role in the differentiation of epithelial cells, such as EMCs in teleosts and keratinocytes in terrestrial vertebrates. After induction of agr2 + EMCs by Delta/Jagged-mediated activation of Notch1a/3, the agr2 + EMCs population is maintained by proliferation (Fig 6A).High proliferation rates (16.8-20%) were detected in 26 s and 24 hpf embryos, while a lower (4.4%)proliferation rate was observed at 48 hpf.Likewise, after specification by Dlc-Notch1a/3 lateral inhibition during late gastrulation and early somite stages, ionocytes also maintain their population by proliferation; a similarly low (3%) proliferation rate was observed in NaR cells at 120 hpf [37].In contrast, mouse Notch1 coordinates keratinocyte growth arrest and differentiation by induction of p21 WAF1/Cip1 expression and early differentiation markers (keratin 14, integrin β1 and β4) [8]. Although EMCs and intestinal goblet cells both express agr2 and serve primarily as secretory cells, their differentiation and regulation are quite different.In zebrafish intestine, Delta-Notch signaling controls the decision between secretory and absorptive cell fates [38].Most intestinal cells adopt a secretory cell fate in DeltaD-defective aei mutants and mib mutant fish embryos.On the contrary, we observed reduced agr2 + EMC numbers in various mutants or morphants defective in Dlc and Jagged ligands (jag1a/1b/2a) or Notch receptors (notch1a/3), and mib mutants (Figs 2, 3, 4, andS5).We suspect that the choice between cell fate specification through Delta-Notch lateral inhibition or Delta/Jagged-induced Notch signaling may determine the distribution patterns and abundances of goblet and mucous cells in the intestine or epidermis.Overall, our findings reveal that the differentiation of agr2 + EMCs is controlled by Delta/Jagged ligand induction of Notch signaling.This result is consistent with a recent publication that showed Notch signaling induces a secretory cell fate change in epidermal multiciliated cells of Xenopus embryos [39].Thus, our results suggest that the specification of three major cell types in the epidermis depends on temporally distinct Notch-dependent signaling events, and this consistent involvement of Notch signaling in epidermal cell development may be common to many vertebrate species. ", "section_name": "Discussion", "section_num": null }, { "section_content": "", "section_name": "Materials and methods", "section_num": null }, { "section_content": "All animal procedures were approved by the Academia Sinica Institutional Animal Care & Use Committee (AS IACUC; Protocol ID: 15-12-918 and 18-12-1250) or by the Institutional Animal Care and Use Committee, NHRI, Taiwan (NHRI-IACUC-106063-A, NHRI-IACUC-107094-A and NHRI-IACUC-109036-A).All animal experiments were carried out in accordance with the approved protocols. ", "section_name": "Ethics statement", "section_num": null }, { "section_content": "Adult zebrafish were maintained in a high-density self-circulation system (Aqua Blue) or in 20-liter aquaria supplied with aerated filtered fresh water, located in a room with a 14/10-h light/dark photoperiod.Embryos were incubated at 28.5˚C or 25˚C depending on the stage at which they were harvested.Stage determination was based on described criteria [40].Various mutants, transgenic lines and wild-types used in this study were: dlc tm98/+ , mib ta52b/+ , mib tfi91/+ , jag1a sa168/+ , jag1b jj59/+ , jag2a sa10076/+ , jag2b sa1720/+ , notch1a th35b/+ , Tg(-6.0k agr2:EGFP) as11Tg , AB wild types, and ASAB wild types. ", "section_name": "Zebrafish strains and breeding conditions", "section_num": null }, { "section_content": "Various morpholio oligonucleotides (MOs) from Gene Tools used in this study were as follows: dlc MO: 5'-CGATAGCAGACTGTGAGAGTAGTCC -3', 4.9 ng [31]; foxi3a ATGMO: 5'-AGACTGTGGAACAAATGATGTCATG -3', 4.3 ng [14]; foxi3a misMO: 5'-AGAGTGTGC AAGAAATCATGTGATG-3', 4.3 ng (mismatched sequence is italized) [14]; grhl1 ATGMO: 5'-GTGACATCTCTTATGGTCGAACTGG-3', 3 ng [21]; grhl1 SPMO: 5'-CTTTGATGAGAG CTTCACCTTTTGT-3', 3 ng [21]; jag1a UTRMO: 5'-CGGTTTGTCTGTCTGTGTGT CTGTC-3', 0.5 ng [41]; jag1a SPMO: 5'-ATAAAAGATACTCACTGCCAGTGCT-3', 1.0 ng; jag1b UTRMO: 5'-TCACGGCTCTAATGTACTCCCCGAT-3', 1.2 ng [42]; jag2a MO: 5'-CTCCAAAATAGTTATGCATGACTCC-3', 4 ng (start codon is underlined); notch3 UTRMO: 5'-ACATCCTTTAAGAAATGAATCGGCG-3', 0.8 ng [43]; notch3 SPMO: 5'-AAGGATCAGTCATCTTACCTTCGCT-3', 2 ng [43] Various MOs were diluted in Danieau solution and individually injected into the cytoplasm of one-or two-cell zygotes. Capped full-length dlc, jag1a, jag1b, jag2a, jag2b, LacZ, notch1a ICD, notch3 ICD, and X-Su (H)/ANK mRNA were synthesized using NotI-linearied PCS2 + -MT-dlc plasmid, NotI-linearized pCS2 + -MT-jag1a plasmid, NotI-linearized pCS2 + -MT-jag1b plasmid, NotI-linearized pCS2 + -jag2a plasmid, SacII-linearized pCS2 + -jag2b plasmid, NotI-linearized pcDNA3.1/Myc-His(+)/LacZplasmid, NotI-linearized pCS2 + -MT-notch1a ICD plasmid, NotI-linearized pCS3 + -MT-notch3 ICD plasmid, and NotI-linearized pCS2 + -X-Su(H)-ANK plasmid as templates with the mMESSAGE mMACHINE SP6 or T7 transcription kit (Invitrogen).Digoxigenin (DIG)-or fluorescein-labeled RNA probes were generated using respective linearized constructs as templates and DIG or fluorescein RNA labeling kit with SP6 or T7 RNA polymerase (SP6, Roche; T7, Invitrogen).Restriction enzymes used for linearization and RNA polymerase for probe synthesis were: agr2 (NcoI/SP6), atp6v1aa (SacII/SP6), dlc (NcoI/SP6), egfp (HindIII/SP6), grhl1 (SalI/T7), jag1a (NcoI/SP6), jag1b (SalI/T7), jag2a (SalI/T7), mucin 5.1(NcoI/SP6), mucin 5.2 (NcoI/SP6) and pvalb8 (SalI/T7).Overexpression experiments were conducted by microinjecting 92 or 184 pg dlc, 250 pg jag1a, 300 pg jag1b, 200 pg jag2a, 500 pg jag2b, 200 pg notch1a ICD, notch3 ICD (10, 50, 100, 200 pg) or 10 pg X-Su(H)-ANK into the cytoplasm of one-or two-cell zygotes. ", "section_name": "Morpholino knockdown, capped mRNA and probe synthesis and mRNA overexpression", "section_num": null }, { "section_content": "Working solutions were prepared from 10 mM MK-0752 (Selleckchem) stock solution and 100% DMSO (Invitrogen) in 1x E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 ).Embryos (20 embryos/mL) were incubated with 100 μM MK-0752 or 1% DMSO from bud to 17 or 20 hpf at 28.5˚C, followed by 1x E3 medium washes and maintenance at 28.5˚C.Embryos were maintained until 24 hpf, at which point they were fixed with 4% paraformaldehyde (PFA) in 1x PBS at 4˚C overnight.Embryos were also incubated with 100 μM MK-0752 or 1% DMSO from bud to 24 hpf at 28.5˚C before fixation.Embryos were incubated with 50 μM MK-0752 or 0.5% DMSO from bud to 13 or 14 hpf, or from 11 to 14 or 15 hpf, followed by 1x E3 medium washes and maintained at 28.5˚C until 24 hpf stage for fixation.Embryos were also incubated with 75 μM MK-0752 or 0.75% DMSO from bud to 15, 16 or 17 hpf, or from 12 to 17 hpf at 28.5˚C, followed by 1x E3 medium washes; embryos were maintained at 28.5˚C until fixation at the 24 hpf stage. ", "section_name": "MK-0752 treatment", "section_num": null }, { "section_content": "Embryos at 24 s, 23 hpf, 29 hpf or 47 hpf were treated with egg water containing 10 mM BrdU and 15% DMSO in 2 mL tube for 20 min on ice.Samples were then transferred to a 9-cm petri dish and washed with egg water five times, then incubated for 40 min at 28.5˚C before fixation at 26 s, 24 hpf, 30 hpf and 48 hpf with 4% PFA in 1x PBS at 4˚C overnight.After several PBST washes, embryos were dehydrated with 25% methanol/75% PBST, 50% methanol/50% PBST, 75% methanol/25% PBST and 100% methanol for 5 min, three times, and stored in 100% methanol at -20˚C. ", "section_name": "BrdU incorporation", "section_num": null }, { "section_content": "Dechorionated embryos at 24 hpf were fixed with freshly prepared 4% PFA in 1x PBS at 4˚C overnight.Embryos were washed with diethyl pyrocarbonate (DEPC)-treated 1x PBS containing 0.1% Tween 20 (PBST) for 5 min at room temperature (RT), three times, followed by dehydration with 25% methanol/75% DEPC-PBST, 50% methanol/50% DEPC-PBST, 75% methanol/25% DEPC-PBST and 100% methanol and storage in 100% methanol at -20˚C.After rehydration with a methanol/DEPC-PBST series and DEPC-PBST washes, embryos were digested with 10 μg/mL proteinase K (Roche) in DEPC-PBST for 3 min at RT, followed by DEPC-PBST washes and re-fixation with 4% PFA in 1x PBS for 20 min at RT.After several DEPC-PBST washes, embryos were incubated in hybridization buffer (50% formamide, 5x SSC, 0.1% Tween-20, 500 μg/mL yeast tRNA, 50 μg/mL heparin adjusted to pH 5.2 with 2 M citric acid) at 65˚C for 3 h before incubation in probe solution (150 ng RNA probe per 300 μL hybridization buffer) at 65˚C overnight for hybridization.Embryos were washed at 65˚C with pre-heated 25%, 50%, 75% Hyb -buffer (50% formamide, 5x SSC, 0.1% Tween-20) in 2x SSC for 10 min each, 2x SSC for 10 min then 1 h and 0.2x SSC containing 0.1% Tween-20 for 30 min four times at 70˚C.Embryos were then incubated in 1% blocking reagent (Roche) for 3 h at RT before incubation with pre-absorbed anti-digoxygenin (DIG)-alkaline phosphatase antibody (1:4000) in 0.5% blocking solution at 4˚C overnight.After several PBST washes, embryos were equilibrated in freshly made staining buffer (0.1 M Tris-HCl pH 9.5, 50 mM MgCl 2 , 0.1 M NaCl, 0.1% Tween-20) for 5 min three times and later stained with NBT/BCIP-containing staining buffer (3.5 μL of 100 mg/mL NBT in 70% dimethylformamide and 3.5 μL of 50 mg/ mL X-phosphate in dimethylformamide added to 1 mL staining buffer) until the color signal was clearly apparent.Embryos were rinsed with PBST, washed in 100% methanol for 20 min.After several PBST washes, embryos were stored in 4% PFA in 1x PBS at 4˚C before samples were photographed. ", "section_name": "Whole-mount in situ hybridization", "section_num": null }, { "section_content": "Dechorinated 48 hpf Tg(-6.0kagr2:EGFP) embryos were fixed with 4% PFA in 1x PBS at 4˚C overnight.After four washes with PBST for 5 min at RT, embryos were dehydrated with 25% methanol/75% PBST, 50% methanol/50% PBST, 75% methanol/25% PBST and 100% methanol for 5 min, three times, and stored in 100% methanol at -20˚C.Embryos were treated with acetone for 15 min at -20˚C before being rehydrated through 75% methanol/25% PBST, 50% methanol/50% PBST, 25% methanol/75% PBST and PBST four times for 5 min each.Embryos were treated with 150 mM Tris-HCl, pH 9.5 for 15 min at 70˚C, followed by re-fixation with 4% PFA in 1x PBS for 5-10 min at RT.After four PBST washes for 5 min each, embryos were blocked in PBST containing 1% blocking reagent for 1.5 h at RT.Embryos were then treated with anti-salmon Agr2 antibody (1:200) and anti-GFP antibody (1:400, Sigma-Aldrich) in PBST containing 1% blocking reagent at 4˚C overnight.After PBST washes for 10 min, six times, embryos were blocked with 1% blocking reagent for 1 h at RT followed by incubation with anti-mouse Alexa Fluor 488 antibody (1:200, Invitrogen) and anti-rabbit Alexa Fluor 568 antibody (1:200, Invitrogen).After six PBST washes for 10 min each, embryos were stored in 4% PFA in 1x PBS at 4˚C before being photographed. ", "section_name": "Double immunofluorescence for Agr2 and EGFP", "section_num": null }, { "section_content": "Dechorinated Tg(-6.0kagr2:EGFP) embryos at 24 or 48 hpf and dechorinated wild-type embryos at 22 or 24 hpf were fixed with 4% PFA in 1x PBS at 4˚C overnight.After four DEPC-PBST washes for 5 min each, embryos were dehydrated through 25% methanol/75% DEPC-PBST, 50% methanol/50% DEPC-PBST, 75% methanol/25% DEPC-PBST and 100% methanol and stored in 100% methanol at -20˚C.After rehydration with a methanol/ DEPC-PBST series and DEPC-PBST washes, embryos were digested with 10 μg/mL proteinase K in DEPC-PBST (1-3 min for 22 hpf, 3 min for 24 hpf and 20 min for 48 hpf) at RT, followed by DEPC-PBST washes and re-fixation with 4% PFA in 1x PBS for 20 min at RT.After several DEPC-PBST washes, embryos were incubated in hybridization buffer (50% formamide, 5x SSC, 0.1% Tween-20, 500 μg/mL yeast tRNA, 50 μg/mL heparin adjusted to pH 5.2 with 2 M citric acid) at 65˚C for 1 h before incubation in probe solution (300 ng each for muc5.1 and muc5.2,300 ng pvalb8 and 150 ng eGFP RNA probes, 150 ng agr2 and 150 ng foxi3a or 150 ng grhl1 in 300 μL hybridization buffer) at 65˚C overnight for hybridization.Embryos were washed with pre-heated 75%, 50%, 25% Hyb -buffer in 2x SSC for 10 min each, 2x SSC for 30 min, two times, and 0.2x SSC for 30 min four times at 70˚C.After two PBST washes for 5 min each, embryos were blocked in 1% blocking reagent in PBST for 1.5 h at RT, followed by incubation with anti-DIG-horse-radish peroxidase (POD) antibody (1:600) in 0.5% blocking reagent at 4˚C overnight.After six PBST washes for 15 min each, embryos were incubated for 45 to 60 min at RT with TSA plus Cy3-tyramide substrate (1:100, Akoya Biosciences) diluted in amplification buffer.Embryos then washed in PBST, 25% PBST/75% methanol, 50% PBST/50% methanol, 25% PBST/75% methanol and 100% methanol for 10 min each at RT, followed by inactivation of POD with 1% H 2 O 2 in 100% methanol for 30 min at RT.Embryos were then blocked with 1% blocking reagent in PBST for 1 h at RT, followed by incubation with anti-fluorescein-POD antibody (1:600) in 0.5% blocking reagent at 4˚C overnight.Embryos were washed with PBST for 15 min six times, followed by incubation in TSA plus FITC-tyramide substrate (1:100) diluted in amplification buffer for 45 to 60 min at RT.Embryos were washed with PBST for 10 min four times and stored in 4% PFA in 1x PBS at 4˚C. ", "section_name": "Double fluorescence in situ hybridization for egfp and muc5.1/5.2, egfp and pvalb8, agr2 and foxi3a or agr2 and grhl1", "section_num": null }, { "section_content": "Dechorinated wild-type embryos at 5 s, 10 s or 22 hpf were fixed with 4% PFA in 1x PBS at 4˚C overnight.After four DEPC-PBST washes for 5 min each, embryos were dehydrated through 25% methanol/75% DEPC-PBST, 50% methanol/50% DEPC-PBST, 75% methanol/ 25% DEPC-PBST and 100% methanol and stored in 100% methanol at -20˚C.After rehydration with a methanol/DEPC-PBST series and DEPC-PBST washes, embryos were digested with 10 μg/mL proteinase K in DEPC-PBST for 1 min at RT, followed by DEPC-PBST washes and re-fixation with 4% PFA in 1x PBS for 20 min at RT.After several DEPC-PBST washes, embryos were incubated in hybridization buffer (50% formamide, 5x SSC, 0.1% Tween-20, 500 μg/mL yeast tRNA, 50 μg/mL heparin adjusted to pH 5.2 with 2 M citric acid) at 65˚C for 4 h before incubation in probe solution (200 ng each for jag1a, jag1b and jag2a and 150 ng dlc, 150 ng foxi3a or 150 ng agr2 in 300 μL hybridization buffer) at 65˚C overnight for hybridization.Embryos were washed with pre-heated 75%, 50%, 25% Hyb -buffer in 2x SSC for 10 min each, 2x SSC for 30 min, two times, and 0.2x SSC for 30 min four times at 65˚C.After two PBST washes for 5 min each, embryos were blocked in 1% blocking reagent in 1x PBST for 3 h at RT, followed by incubation with anti-DIG-POD antibody (1:600) in 0.5% blocking reagent at 4˚C overnight.After six to eight PBST washes for 15 min each, embryos were incubated for 40 min at RT with TSA plus Cy3-tyramide substrate (1:100, Akoya Biosciences) diluted in amplification buffer.Embryos were washed for 10 min three times in PBST and incubated in 100 mM glycin pH 2.2 for 10 min at RT.Embryos were washed for 10 min each in 50% methanol/50% PBST and 100% methanol at RT, followed by PBST washes for 10 min two times.Embryos were blocked in 1% blocking reagent in PBST for 1 h at RT, followed by incubation with anti-fluorescein-POD antibody (1:600) in 0.5% blocking reagent at 4˚C overnight.Embryos were washed with PBST for 15 min six to eight times, followed by incubation in TSA plus FITC-tyramide substrate (1:100) diluted in amplification buffer for 40 min at RT.Embryos were washed with PBST for 10 min four times and stored in 4% PFA in 1x PBS at 4˚C. ", "section_name": "Double fluorescence in situ hybridization for jag1a/jag1b/jag2a and foxi3a, grhl1, dlc or agr2", "section_num": null }, { "section_content": "Dechorinated wild-type embryos at 5 s were fixed with 4% PFA in 1x PBS at 4˚C overnight.After four DEPC-PBST washes for 5 min each, embryos were dehydrated through 25% methanol/75% DEPC-PBST, 50% methanol/50% DEPC-PBST, 75% methanol/25% DEPC-PBST and 100% methanol and stored in 100% methanol at -20˚C.After rehydration with a methanol/ DEPC-PBST series and DEPC-PBST washes, embryos were digested with 10 μg/mL proteinase K in DEPC-PBST for 1 min at RT, followed by DEPC-PBST washes and re-fixation with 4% PFA in 1x PBS for 20 min at RT.After several DEPC-PBST washes, embryos were incubated in hybridization buffer (50% formamide, 5x SSC, 0.1% Tween-20, 500 μg/mL yeast tRNA, 50 μg/mL heparin adjusted to pH 5.2 with 2 M citric acid) at 65˚C for 4 h before incubation in probe solution (200 ng each for jag1a, jag1b and jag2a in 300 μL hybridization buffer) at 65˚C overnight for hybridization.Embryos were washed with pre-heated 75%, 50%, 25% Hyb - buffer in 2x SSC for 10 min each, 2x SSC for 30 min, two times, and 0.2x SSC for 30 min four times at 65˚C.After two PBST washes for 5 min each, embryos were blocked in 1% blocking reagent in PBST for 3 h at RT, followed by incubation with anti-DIG-POD antibody (1:600) in 0.5% blocking reagent at 4˚C overnight.After six to eight PBST washes for 15 min each, embryos were incubated for 40 min at RT with TSA plus Cy3-tyramide substrate (1:100, Akoya Biosciences) diluted in amplification buffer.Embryos were washed for 10 min three times in PBST, followed by incubation in 100 mM glycine pH 2.2 for 10 min.Embryos were washed for 10 min each in 50% methanol/50% PBST and 100% methanol at RT, followed by PBST washes for 10 min two times.Embryos were treated with 150 mM Tris-HCl, pH 9.5 for 15 min at 70˚C, followed by re-fixation with 4% PFA in 1x PBS for 5-10 min at RT.After four PBST washes for 5 min each, embryos were blocked in PBST containing 0.5% blocking reagent for 1 h at RT.Embryos then treated with anti-p63 antibody (1:50, Abcam) in PBST containing 0.5% blocking reagent at 4˚C overnight.After PBST washes for 10 min, six times, embryos were blocked with 1% blocking reagent for 1 h at RT followed by incubation with anti-mouse Alexa Fluor 488 antibody (1:200, Invitrogen).After PBST washes for 10 min, six times, embryos were stored in 4% PFA in 1x PBS at 4˚C. ", "section_name": "Imunofluorescence for p63 and fluorescence in situ hybridization for jag1a, jag1b or jag2a", "section_num": null }, { "section_content": "The donor embryos were injected with 5% dextran-Alexa Fluor 488 (anionic, fixable, 10 kDa) in 0.1 M KCl at the 1-cell stage.To obtain single cells for transplantation, cells from the ventral ectoderm at shield stage were loaded into a transplant needle (10 psi) and released on a 1.5% agarose plate containing PBS.Single cells were then loaded into a needle and transferred into the ventral ectoderm region of host embryos.Dechorinated 24 hpf recipients were fixed with 4% PFA at 4˚C overnight.For Agr2 + cells and keratinocytes, fixed embryos were washed with 1x PBS containing 0.3% triton X-100 (PBT) for 5 min three times at RT.After washing with PBS for 5 min twice, embryos were treated with 10 μg/mL proteinase K for 5 min at 28˚C.Embryos were then washed twice with PBT for 5 min each, and further fixed with 4% PFA for 20 min at RT.After washing with PBT for 5 min three times, embryos were blocked with 4% BSA in PBT for 1 h at RT.Embryos were incubated with rabbit anti-salmon Agr2 antibody (1:200) and mouse anti-human P63 antibody (1:200; 4A4, Abcam) in 4% BSA at 4˚C overnight.After washing with PBT for 15 min four times, embryos were incubated with goat antirabbit IgG Alexa Fluor 647 antibody (1:200) and goat anti-mouse IgG Alexa Fluor 594 plus (1:200) in 4% BSA at 4˚C overnight.For experiments on Agr2 + cells and ionocytes, fixed embryos were dehydrated with 25% methanol/75% PBST, 50% methanol/50% PBST, 75% methanol/25% PBST and 100% methanol for 10 min two times and stored in 100% methanol at -20˚C.Embryos were treated with 100% acetone for 15 min at -20˚C before being rehydrated with 75% methanol/25% PBST, 50% methanol/50% PBST, 25% methanol/75% PBST and 100% PBT for 5 min four times.Embryos were blocked with 4% BSA in PBT for 1 h at RT.Embryos were incubated with rabbit anti-salmon Agr2 antibody (1:200) and mouse antichicken alpha-subunit of Na + , K + -ATPase antibody (1:200; α5, DSHB Hybridoma) in 4% BSA at 4˚C overnight.After washing with PBT for 15 min four times, embryos were incubated with goat anti-rabbit IgG Alexa Fluor 647 antibody (1:200) and goat anti-mouse IgG Alexa Fluor 594 plus (1:200) in 4% BSA at 4˚C overnight.After washing with PBT for 15 min four times, embryos were washed with PBS for 5 min three times.Embryos were then stored in antifade reagent (SlowFade Diamond Antifade Mountant with DAPI). ", "section_name": "Lineage tracing experiments", "section_num": null }, { "section_content": "Fixed BrdU-incorporated embryos at 30 and 48 hpf were rehydrated with 75% methanol/25% PBST, 50% methanol/50% PBST, 25% methanol/75% PBST and PBST for 5 min four times.After treatment of 10 μg/mL proteinase K (3 min for 30 hpf and 20 min for 48 hpf) at RT, embryos were washed with PBST for 5-10 min four times, followed by a brief rinse with 2 N HCl and incubation in 2 N HCl for 20 min at RT.After four PBST washes for 5-10 min each, embryos were blocked in 1% blocking reagent for 1 h at RT before incubation with mouse anti-BrdU antibody (1:200, BD Biosciences) and rabbit anti-salmon Agr2 antibody (1:200) in 1% blocking reagents at 4˚C overnight.Following six PBST washes for 15 min each, embryos were blocked in 1% blocking reagent for 1 h at RT before incubation with anti-mouse Alexa Fluor 488 (1:200) and anti-rabbit Alexa Fluor 568 (1:200) in 0.5% blocking reagent for 3 h at RT.Embryos were then washed with 1x PBS containing 0.3% Tween 20 for 15 min twice, followed by four PBST washes for 15 min each.Embryos were incubated in Hoechst 33342 in PBST (Invitrogen) for 30 min at RT.After several PBST washes, embryos were stored in 4% PFA in 1x PBS at 4˚C before being embedded in 1% low-melting agar for confocal imaging. ", "section_name": "Double immunofluorescence for BrdU and Agr2", "section_num": null }, { "section_content": "BrdU-treated embryos fixed at 26 s and 24 hpf were rehydrated with 75% methanol/25% DEPC-PBST, 50% methanol/50% DEPC-PBST, 25% methanol/75% DEPC-PBST and washed with DEPC-PBST for 5 min four times.After incubation in Hyb -buffer for 5 min and in hybridization buffer for 1 h at 65˚C, embryos were hybridized with agr2 antisense RNA probe (150 ng in 300 μL) in hybridization buffer at 65˚C overnight.Embryos were washed at 65˚C with pre-heated 25%, 50%, 75% Hyb -buffer in 2x SSC for 10 min each, 2x SSC for 10 min then 1 h, and 0.2x SSC containing 0.1% Tween-20 for 30 min, four times, at 70˚C.After two PBST washes for 5 min each, embryos were blocked in 1% blocking reagent in PBST for 1.5 h at RT before incubation with anti-DIG-POD antibody (1:600) in 0.5% blocking reagent in PBST at 4˚C overnight.Following six PBST washes for 15 min, embryos were incubated in TSA plus Cy3-tyramide substrate (1:100 diluted in amplification buffer) for 45-60 min at RT.Embryos were then washed with PBST for 10 min twice at RT, followed by incubation in 100 mM glycine for 20 min at RT.After PBST washes for 5 min, five times at RT, embryos were treated with 2 N HCl for 20 min at RT.After PBST washes for 5 min, five times, at RT, embryos were incubated in 1% blocking reagent for 1 h at RT.Embryos were then incubated with anti-BrdU antibody (1:200) in 1% blocking reagent at 4˚C overnight.Embryos were washed with PBST for 10 min, six times, followed by incubation with anti-mouse Alexa Fluor 488 (1:200) in 0.5% blocking reagent for 3 h at RT.After six PBST washes for 10 min each, embryos were incubated in Hoechst 33342 in PBST (1:1000) for 30 min at RT, followed by PBST washes.Embryos were stored in 4% PFA in 1 x PBS at 4˚C before being embedded in 1% low-melting agar for confocal imaging. ", "section_name": "BrdU immunofluorescence and agr2 fluorescence in situ hybridization", "section_num": null }, { "section_content": "Tg(-6.0k agr2:EGFP) embryos at 72 hpf were fixed in prefixative solution (4% PFA and 2.5% glutaraldehyde in 1 x PBS, pH 7.2) at 4˚C overnight.Samples were washed with 1x PBS, pH 7.2 for 15 min, three times, at RT with shaking, followed by incubation in postfixative solution (1-2% osmium tetroxide in 1x PBS, pH 7.2) for 2 h at RT in the dark.Samples were washed with 1x PBS for 15 min, two times, at RT with shaking in the dark, followed by dehydration in 30% ethanol/1 x PBS, 50% ethanol/50% 1 x PBS, 70% ethanol/30% 1 x PBS, 80% ethanol/20% 1 x PBS, 90% ethanol/10% 1 x PBS, 95% ethanol/5% 1 x PBS and 100% ethanol for 15 min at RT with shaking in the dark.Samples were dehydrated in 100% ethanol for 15 min, three times, followed by incubation in 100% propylene oxide for 15 min, three times, at RT with shaking in the dark.Samples were incubated in solution containing propylene oxide and Spurr's resin (3:1) at RT overnight, followed by incubation in a solution containing propylene oxide and Spurr's resin (1:1) and a solution containing propylene oxide and Spurr's resin (1:3) for 8 h at RT.Samples were then embedded in 100% Spurr's resin for 8 h, three times, at RT with shaking, followed by embedding in 100% Spurr's resin at 60˚C for 24 h.Sections (90 nm) were produced using a Leica EM UC7 ultramicrotome and examined using an electron microscope (Tecnai G2 F20S-TWIN, FEI) equipped with a digital camera (UltraScan 1000, Gatan). For immuno-EM sample preparation [44], embryos were anaesthetized and pre-fixed by 4% PFA for 30 min.Embryos were incubated in 20% BSA at RT and placed into HPF carrier A (200 μm in depth) and carrier B as cap (flat side) to process high-pressure freezing of embryos in EM HPF (HPM100, Leica) to -196˚C.Embryos were then transferred to EM AFS2 (Leica) for freeze substitution: Embryos were incubated in 0.2% uranyl acetate in acetone containing 5% water and 4% methanol for 2 to 3 days at -90˚C; embryos were warmed to -45˚C through a 5˚C per day gradient; Embryos were washed with acetone, followed by infiltration with gradient Lowicryl resin (HM20) over one day at -45˚C; Embryos were polymerized using UV light for 2 days at -25˚C, followed by 2 days at 20˚C.For immunogold labeling of GFP, sections (90 nm) were washed with 1 x PBS for 10 min three times, followed by blocking in 2% BSA/1 x PBS for 30 min and incubated with anti-GFP (1:200 or 1:500; Sigma-Aldrich) diluted in 1% BSA/1 x PBS for overnight.After PBS washes for 10 min three times, sections were incubated with Protein A conjugated with 5 nm gold (1: 50; BBI) diluted in PBS for 2 h.After PBS washes for 10 min three times, sections were fixed in 1% glutaraldehyde for 10 min and washed with dH 2 O for 1 min before imaging on an electron microscope (Tecnai G2 F20S-TWIN, FEI) equipped with a digital camera (UltraScan 1000, Gatan). ", "section_name": "Transmission electron microscopy and immunogold labeling", "section_num": null }, { "section_content": "Images of embryos were taken using an AxioCam HRC camera on a Zeiss Axioplan 2 microscope in DIC mode.High-resolution fluorescence images were taken using a Leica TCS-SP5-MP, a Zeiss LSM880 with AiryScan or a Nikon A1R confocal microscope.EMC number was counted from images taken on compound or confocal microscopes using ImageJ software as follows: (i) the image was loaded into ImageJ; (ii) 'Cell counter' was selected from 'Analyze' items on the Plugins menu; (iii) 'Initialize' was selected; and (iv) cell number was determined.All experimental values are presented as mean ± SEM.Two-tailed Student's t-test with unequal variance was conducted in Microsoft Excel to compare experimental groups. ", "section_name": "Photography, quantification of epidermal mucous cell number and statistical methods", "section_num": null }, { "section_content": "DNase I-treated total RNA (1 μg) isolated from AB or jag1a mutant embryos at 24 hpf was used to produce first-strand cDNA using oligo (dT) primer and SuperScript III Reverse transcriptase (Invitrogen) at 65˚C for 5 min and 50˚C for 1 h.qPCRs were conducted by adding cDNAs into 1x SYBR green PCR master mix containing 5 pmole of the respective forward and reverse primers for 18s rRNA, jag1a, jag1b, jag2a or jag2b genes.PCR condition were set to 94˚C for 1 min for 1 cycle; and 94˚C for 10 s, 60˚C for 10 s and 72˚C for 10 s for 40 cycles; 72˚C for 1 min.Respective primer pair for 18s rRNA was F-TCGCTAGTTGGCATCGTTTATG and R-CGGAGGTTCGAAGACGATCA; for jag1a was F-CAGGCTGGTCCGGGTTATTT and R-GGATGAACTGCGACCAGGAA; for jag1b was F-GCTGTGCGGAAACTCTCTCT and R-GCAGCGAAATGCTAACCGAC; for jag2a was F-CAGCAGCACAGGACACACTA and R-GCCTTGGGCCATTCGGATTA; for jag2b was F-TTCACGAGGAGGGCTAGAGT and R-CTGGAGACCCTGAAACCGAG.To calculate the expression level, the comparative cycle threshold (Ct) was used (Roche).4Ct = avg.Ct GOI -avg.Ct 18s rRNA ; 44Ct = 4Ct jag mutant -4Ct wt ; Fold change = 2 ( -44Ct ); The biological variance was reported as the SEM, calculated as the standard deviation of multiple samples from two repeats divided by the square root of the number of samples from two repeats. ", "section_name": "Real-time quantitative reverse-transcription PCR (RT-qPCR)", "section_num": null } ]	[ { "section_content": "We thank professors R.N. Morrison and B. F. Nowak for providing the anti-salmon Agr2 antiserum and Professor P. P. Hwang for providing anti-Na + -K + -ATPase (α5) antibody.We are grateful to the Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica for transmission electron microscope and confocal assistance.We also thank the Taiwan Zebrafish Core Facilities (TZCAS and TZeNH), which are supported by the Ministry of Science and Technology, Taiwan [MOST 110-2740-B-400-001], for providing the ASAB and AB strains. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "This research was supported by the Ministry of Science and Technology, Taiwan [MOST 105-2313-B-001-005-MY3 (SPLH); MOST 108-2313-B-001-007-MY3 (SPLH); MOST 106-2311-B-400-003-MY3 (YJJ); MOST 110-2311-B-400-001 (YJJ)], ICOB intramural funding (SPLH) and NHRI intramural funding (YJJ).The funders had no role in study design, data collection and ", "section_name": "", "section_num": "" }, { "section_content": "All relevant data are within the manuscript and its Supporting Information files. ", "section_name": "", "section_num": "" }, { "section_content": ". Similar agr2 + EMC numbers were observed in the trunks and yolks of jag1a homozygous mutants compared to sibling wild-type embryos at 24 hpf.Scale bars, 100 μm.(B).Jag1a mutant protein is predicted to contain only MNNL, DSL and six EGF domains with a premature stop codon.However, similar mRNA expression levels for jag1a, jag1b, jag2a and jag2b were detected in both jag1a homozygous mutants and wildtype embryos by RT-qPCR, using 18s rRNA as a reference gene.(C).Similar reduced levels of trunk and yolk agr2 + EMC numbers were detected in wild-types or jag1a mutants injected with both jag1b and jag2a MOs at 24 hpf.(D).Similar agr2 + EMC numbers in the trunks and yolks were detected in embryos injected with 500 pg jag2b mRNA compared to LacZ-injected embryos at 24 hpf.No alterations of agr2 + EMC numbers in the trunks and yolks can be identified among jag2b heterozygous, homozygous mutant or sibling wild-type embryos at 24 hpf.(E).Significant reductions of pvalb8-positive cell numbers were detected in the trunks of jag2a mutants or embryos injected with jag1a SPMO or jag1b UTRMO.Substantial decreases in pvalb8-positive cell numbers were observed in the trunks and yolks of embryos injected with jag1a UTRMO at 24 hpf.Arrows, EMCs or pvalb8-positive cells.Scale bars, 100 μm.Mean ± SEM.Student's t-test.� p<0.05; �� p<0.01; �� p<0.001; ns, not significant.Underlying data are available in S2 Data.(TIF) Comparable trunk and yolk agr2 + EMC numbers were detected in wild-types and embryos injected with foxi3a ATGMO at 24 hpf.Low but significant increases in trunk agr2 + EMC numbers were observed in embryos injected with foxi3a ATGMO compared with foxi3a misMOinjected control embryos.atp6v1aa-positive ionocytes were not detected in embryos injected with foxi3a ATGMO unlike those injected with control foxi3a misMO and wild-types.(B).Comparable trunk and yolk agr2 + EMC numbers were detected in wild-types and embryos injected with grhl1 ATGMO or grhl1 SPMO at 24 hpf.Increased grhl1-positive cell numbers were observed in the trunks and yolks of embryos injected with grhl1 ATGMO or grhl1 SPMO compared with wild-types.(C).Co-expression of dlc and jag1a, jag1b or jag2a was detected in embryos at 5 s and 10 s. White box indicates the enlarged area.White arrows indicate example cells with colocalization of markers.Yellow arrows indicate example jag1a-, jag1b-or jag2aexpressing cells.(D).Similar trunk and yolk agr2 + EMC numbers were detected in sibling wild types and dlc tm98 mutants at 24 hpf.Significant reductions in the numbers of trunk and total agr2 + EMCs were detected in dlc morphants compared to wild-type embryos at 24 hpf.Increased foxi3a + ionocyte numbers were observed in the yolk sac of dlc morphants compared to wild types at bud.Increases in the numbers of trunk or yolk and total agr2 + EMCs were detected in 92 or 184 pg dlc mRNA-injected embryos compared to LacZ mRNA-injected embryos at 24 hpf.Similar trunk and yolk pvalb8-positive cell numbers were observed in 92 or 184 pg dlc mRNA-injected embryos and LacZ mRNA-injected embryos at 24 hpf.Reductions in trunk and yolk agr2 + EMC numbers were detected in embryos injected with dlc, jag1a, jag1b and jag2a MOs compared with wild-type embryos at 24 hpf.Arrows, EMCs or pvalb8positive cells.Scale bars, 100 μm.Mean ± SEM.Student's t-test.� p<0.05; �� p<0.01; �� p<0.001; ns, not significant.Underlying data are available in S2 Data.(TIF) S1 Data.Data underlying Figs 2Ac, 2Af, 2Bf, 3Ac, 3Af, 3Bg, 3Bh, 3Bi, 3Bl, 4Ac, 4Af, 4Ai, 4Bc, 4Bd, 4Bg, 4Bh, 4Bk, 4Bl, 4Ce and6Am Conceptualization: Yu-Fen Lu, Da-Wei Liu, I-Chen Li, Yun-Jin Jiang, Sheng-Ping L. Hwang. Data curation: Yu-Fen Lu, Da-Wei Liu, I-Chen Li, Jamie Lin, Chien-Ming Wang, Kuo-Chang Chu, Hsiao-Hui Kuo, Che-Yi Lin. Funding acquisition: Yun-Jin Jiang, Sheng-Ping L. Hwang. Investigation: Yu-Fen Lu, Da-Wei Liu, I-Chen Li, Jamie Lin, Chien-Ming Wang, Kuo-Chang Chu, Hsiao-Hui Kuo, Che-Yi Lin. Methodology: Yu-Fen Lu, Da-Wei Liu, I-Chen Li, Jamie Lin, Chien-Ming Wang, Kuo-Chang Chu, Hsiao-Hui Kuo, Che-Yi Lin, Ling-Huei Yih. Resources: Ling-Huei Yih, Yun-Jin Jiang, Sheng-Ping L. Hwang. Software: Che-Yi Lin. Supervision: Yun-Jin Jiang, Sheng-Ping L. Hwang. Validation: Yu-Fen Lu, Da-Wei Liu, I-Chen Li, Jamie Lin, Chien-Ming Wang, Kuo-Chang Chu, Hsiao-Hui Kuo, Ling-Huei Yih, Yun-Jin Jiang, Sheng-Ping L. Hwang. Writing -original draft: Yun-Jin Jiang, Sheng-Ping L. Hwang. Yu-Fen Lu, Da-Wei Liu, Ling-Huei Yih, Yun-Jin Jiang, Sheng-Ping L. Hwang. ", "section_name": "S5 Fig. Expression pattern of dlc and jag1a/jag1b/jag2a and Dlc but not ionocyte or grhl1positive cells participate in the development of agr2 + epidermal mucous cells. (A).", "section_num": null }, { "section_content": ". Similar agr2 + EMC numbers were observed in the trunks and yolks of jag1a homozygous mutants compared to sibling wild-type embryos at 24 hpf.Scale bars, 100 μm.(B).Jag1a mutant protein is predicted to contain only MNNL, DSL and six EGF domains with a premature stop codon.However, similar mRNA expression levels for jag1a, jag1b, jag2a and jag2b were detected in both jag1a homozygous mutants and wildtype embryos by RT-qPCR, using 18s rRNA as a reference gene.(C).Similar reduced levels of trunk and yolk agr2 + EMC numbers were detected in wild-types or jag1a mutants injected with both jag1b and jag2a MOs at 24 hpf.(D).Similar agr2 + EMC numbers in the trunks and yolks were detected in embryos injected with 500 pg jag2b mRNA compared to LacZ-injected embryos at 24 hpf.No alterations of agr2 + EMC numbers in the trunks and yolks can be identified among jag2b heterozygous, homozygous mutant or sibling wild-type embryos at 24 hpf.(E).Significant reductions of pvalb8-positive cell numbers were detected in the trunks of jag2a mutants or embryos injected with jag1a SPMO or jag1b UTRMO.Substantial decreases in pvalb8-positive cell numbers were observed in the trunks and yolks of embryos injected with jag1a UTRMO at 24 hpf.Arrows, EMCs or pvalb8-positive cells.Scale bars, 100 μm.Mean ± SEM.Student's t-test.� p<0.05; �� p<0.01; �� p<0.001; ns, not significant.Underlying data are available in S2 Data.(TIF) ", "section_name": "", "section_num": "" }, { "section_content": "Comparable trunk and yolk agr2 + EMC numbers were detected in wild-types and embryos injected with foxi3a ATGMO at 24 hpf.Low but significant increases in trunk agr2 + EMC numbers were observed in embryos injected with foxi3a ATGMO compared with foxi3a misMOinjected control embryos.atp6v1aa-positive ionocytes were not detected in embryos injected with foxi3a ATGMO unlike those injected with control foxi3a misMO and wild-types.(B).Comparable trunk and yolk agr2 + EMC numbers were detected in wild-types and embryos injected with grhl1 ATGMO or grhl1 SPMO at 24 hpf.Increased grhl1-positive cell numbers were observed in the trunks and yolks of embryos injected with grhl1 ATGMO or grhl1 SPMO compared with wild-types.(C).Co-expression of dlc and jag1a, jag1b or jag2a was detected in embryos at 5 s and 10 s. White box indicates the enlarged area.White arrows indicate example cells with colocalization of markers.Yellow arrows indicate example jag1a-, jag1b-or jag2aexpressing cells.(D).Similar trunk and yolk agr2 + EMC numbers were detected in sibling wild types and dlc tm98 mutants at 24 hpf.Significant reductions in the numbers of trunk and total agr2 + EMCs were detected in dlc morphants compared to wild-type embryos at 24 hpf.Increased foxi3a + ionocyte numbers were observed in the yolk sac of dlc morphants compared to wild types at bud.Increases in the numbers of trunk or yolk and total agr2 + EMCs were detected in 92 or 184 pg dlc mRNA-injected embryos compared to LacZ mRNA-injected embryos at 24 hpf.Similar trunk and yolk pvalb8-positive cell numbers were observed in 92 or 184 pg dlc mRNA-injected embryos and LacZ mRNA-injected embryos at 24 hpf.Reductions in trunk and yolk agr2 + EMC numbers were detected in embryos injected with dlc, jag1a, jag1b and jag2a MOs compared with wild-type embryos at 24 hpf.Arrows, EMCs or pvalb8positive cells.Scale bars, 100 μm.Mean ± SEM.Student's t-test.� p<0.05; �� p<0.01; �� p<0.001; ns, not significant.Underlying data are available in S2 Data.(TIF) S1 Data.Data underlying Figs 2Ac, 2Af, 2Bf, 3Ac, 3Af, 3Bg, 3Bh, 3Bi, 3Bl, 4Ac, 4Af, 4Ai, 4Bc, 4Bd, 4Bg, 4Bh, 4Bk, 4Bl, 4Ce and6Am ", "section_name": "S5 Fig. Expression pattern of dlc and jag1a/jag1b/jag2a and Dlc but not ionocyte or grhl1positive cells participate in the development of agr2 + epidermal mucous cells. (A).", "section_num": null }, { "section_content": "Conceptualization: Yu-Fen Lu, Da-Wei Liu, I-Chen Li, Yun-Jin Jiang, Sheng-Ping L. Hwang. Data curation: Yu-Fen Lu, Da-Wei Liu, I-Chen Li, Jamie Lin, Chien-Ming Wang, Kuo-Chang Chu, Hsiao-Hui Kuo, Che-Yi Lin. Funding acquisition: Yun-Jin Jiang, Sheng-Ping L. Hwang. Investigation: Yu-Fen Lu, Da-Wei Liu, I-Chen Li, Jamie Lin, Chien-Ming Wang, Kuo-Chang Chu, Hsiao-Hui Kuo, Che-Yi Lin. Methodology: Yu-Fen Lu, Da-Wei Liu, I-Chen Li, Jamie Lin, Chien-Ming Wang, Kuo-Chang Chu, Hsiao-Hui Kuo, Che-Yi Lin, Ling-Huei Yih. Resources: Ling-Huei Yih, Yun-Jin Jiang, Sheng-Ping L. Hwang. Software: Che-Yi Lin. Supervision: Yun-Jin Jiang, Sheng-Ping L. Hwang. Validation: Yu-Fen Lu, Da-Wei Liu, I-Chen Li, Jamie Lin, Chien-Ming Wang, Kuo-Chang Chu, Hsiao-Hui Kuo, Ling-Huei Yih, Yun-Jin Jiang, Sheng-Ping L. Hwang. Writing -original draft: Yun-Jin Jiang, Sheng-Ping L. Hwang. ", "section_name": "Author Contributions", "section_num": null }, { "section_content": "Yu-Fen Lu, Da-Wei Liu, Ling-Huei Yih, Yun-Jin Jiang, Sheng-Ping L. Hwang. ", "section_name": "Writing -review & editing:", "section_num": null } ]
10.1038/s41416-020-0887-6	UGT2B17 modifies drug response in chronic lymphocytic leukaemia	<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>High UGT2B17 is associated with poor prognosis in untreated chronic lymphocytic leukaemia (CLL) patients and its expression is induced in non-responders to fludarabine-containing regimens. We examined whether UGT2B17, the predominant lymphoid glucuronosyltransferase, affects leukaemic drug response and is involved in the metabolic inactivation of anti-leukaemic agents.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>Functional enzymatic assays and patients’ plasma samples were analysed by mass-spectrometry to evaluate drug inactivation by UGT2B17. Cytotoxicity assays and RNA sequencing were used to assess drug response and transcriptome changes associated with high UGT2B17 levels.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>High UGT2B17 in B-cell models led to reduced sensitivity to fludarabine, ibrutinib and idelalisib. UGT2B17 expression in leukaemic cells involved a non-canonical promoter and was induced by short-term treatment with these anti-leukaemics. Glucuronides of both fludarabine and ibrutinib were detected in CLL patients on respective treatment, however UGT2B17 conjugated fludarabine but not ibrutinib. AMP-activated protein kinase emerges as a pathway associated with high UGT2B17 in fludarabine-treated patients and drug-treated cell models. The expression changes linked to UGT2B17 exposed nuclear factor kappa B as a key regulatory hub.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Data imply that UGT2B17 represents a mechanism altering drug response in CLL through direct inactivation but would also involve additional mechanisms for drugs not inactivated by UGT2B17.</jats:p> </jats:sec>	[ { "section_content": "Chronic lymphocytic leukaemia (CLL) is the most frequent adult leukaemia in the western world.Cancer growth varies widely with indolent and aggressive forms of CLL.In recent years, there has been substantial progress in the clinical management of CLL patients supported by a better risk stratification and the introduction of a number of novel therapeutic agents. 1 These advances significantly improved clinical outcomes of CLL patients. Pre-treatment evaluation of informative prognostic markers helps the stratification of CLL patients into risk subgroups.These markers include unmutated immunoglobulin heavy chain variable region (U-IGHV), del(17p) or TP53 mutations, which are associated with poor response to treatment and predict earlier relapse after achieving initial haematological remission. 2For treatment-naïve patients with these high-risk features, the Bruton tyrosine kinase (BTK) inhibitor ibrutinib is indicated as an initial systemic therapy rather than chemoimmunotherapy. 3,4The latter is based on the purine analogue fludarabine or alkylating agent bendamustine or chlorambucil backbones combined to the monoclonal antibody rituximab directed against the CD20 B-cell marker. 5Other therapeutic small molecules include two distinct mechanisms of actions with venetoclax, a BCL-2 inhibitor, 6 idelalisib, a phosphatidylinositol-3-kinase (PI3K) δ inhibitor, and duvelisib, a dual inhibitor of PI3K δ and γ isoforms. 7,8Phase 3 trials are ongoing for additional agents such as the second-generation oral BTK inhibitor acalabrutinib and the Syk/Jak inhibitor cerdulatinib. 9,10Whilst treatment has significantly advanced patients' survival, it is also associated with incomplete clonal eradication, relapse, refractoriness or transformation to a more aggressive lymphoma (Richter's syndrome). 3oth innate and acquired resistance represent major challenges for long-term disease control and an intense area of research.Findings reveal that targeted agents have unique mechanisms of resistance compared with chemotherapy.Genetic anomalies associated with fludarabine refractoriness include mutations in TP53, SF3B1, NOTCH1 and BIRC3 genes. 11,12Other genetic screens have recently identified further genes that could be implicated in fludarabine sensitivity such as ARID5B and BRAF. 13,14As targeted therapy agents have become more established in cancer therapy, recent reports attribute acquired hypermorphic mutations in genes of targeted pathways, including BTK and its immediate downstream effector phospholipase C, γ2 (PLCG2) critical for BCR signalling. 15Mechanisms of resistance to other targeted agents, so early in clinical use, have not yet been portrayed in CLL patients but work is ongoing in this area. Additional contributing mechanisms of reduced drug sensitivity that may be shared by targeted agents and chemotherapy may include increased drug efflux by ATP-dependent transporters and drug inactivation by metabolising pathways such as the glucuronidation pathway by UDP-glucuronosyltransferase enzymes (UGTs).By conjugating lipophilic drugs to glucuronic acid (GlcA), UGTs impair biological activity and enhance water solubility of drugs, driving their elimination and a lack of drug efficacy. 168][19] However, for most drugs used in CLL, the glucuronidation pathway has not been comprehensively studied.Reports have established that high UGT2B17 expression is an adverse prognostic factor in CLL, associated with shorter treatment-free survival, overall survival and patients requiring more treatment. 17,20,21The link between UGT2B17, adverse CLL features and progression appears complex, and precise molecular mechanisms underlying these observations remain to be elucidated.In cohorts of CLL patients, UGT2B17 was associated with unmutated IgHV and predicted poor survival, 17 and was further associated with progressive disease within the group of patients with a more favourable profile (mutated IgHV). 21A recent study supports that UGT2B17-mediated metabolism would participate in the regulation of signalling pathways critical for CLL progression. 22igh UGT2B17 expression in leukaemic cells was further linked to a lack of response to fludarabine-containing regimens. 17UGT2B17 is one of the 19 functional human drug-conjugating UGT enzymes, also involved in regulating homoeostasis of a number of endogenous metabolites, however its role in conjugating antileukaemics remains largely unknown. 16n the present study, we investigated the response to antileukaemic agents namely fludarabine, ibrutinib, idelalisib, bendamustine, chlorambucil, venetoclax, acalabrutinib, cerdulatinib and duvelisib in relation to UGT2B17 expression levels.We then sought to evaluate the UGT-mediated enzymatic inactivation of drugs relevant to CLL and the possible involvement of the UGT2B17 enzyme.Our data support that high UGT2B17 expression alters drug response by direct inactivation but likely also through other mechanisms.We further evidence that the UGT pathway is involved in the inactivation of the majority of antileukaemic agents used in CLL, suggesting that this pathway may be associated with drug resistance. ", "section_name": "BACKGROUND", "section_num": null }, { "section_content": "", "section_name": "METHODS", "section_num": null }, { "section_content": "Cryopreserved peripheral blood mononuclear cells (PBMCs) and plasma samples were obtained from CLL patients who were diagnosed between 1987 and 2011 at Vienna General Hospital.Patients provided informed consent.The study was carried out in accordance with the Declaration of Helsinki and was approved by the local ethical research committees of the Medical University of Vienna (Ethics vote 1499/2015) and the CHU de Québec (A14-10-1205).UGT2B17 mRNA analysis was performed in CD19 + -sorted cells of twenty CLL patients before and after the first cycle of treatment with fludarabine-containing regimens as described. 23lasma samples for quantification of glucuronides in circulation were available for two patients in the 1st week after the first cycle of treatment with fludarabine-chlorambucil-rituximab and for 15 patients during treatment with ibrutinib.The B lymphoblastoid leukaemic cell lines included p53 wild-type (EHEB, JVM-2) and p53 mutated (MEC-1) cell lines purchased from ATCC (Manassas, VA, USA) or DSMZ (Braunschweig, Germany).Cells overexpressing UGT2B17 were previously described. 22Cell culture components were all purchased from Wisent Bioproducts (Saint-Bruno, QC, Canada). ", "section_name": "Patients, primary CLL cells and cell lines", "section_num": null }, { "section_content": "Glucuronidation assays were conducted using human liver, intestine and kidney microsomes (Xenotech, Kansas City, KS, USA) or supersomes expressing individual human UGT isoforms (Corning, MA, USA).In the absence of commercially available UGT2B11 supersomes, microsomal proteins derived from HEK293 expressing the recombinant UGT2B11 enzyme were used.Substrates included dihydrotestosterone (DHT) purchased from Steraloids (Newport, RI, USA), fludarabine and chlorambucil from Sigma-Aldrich (Oakville, ON, Canada) and bendamustine from Toronto Research Chemicals (North York, ON, Canada).All other compounds were purchased from Selleckchem (Houston, TX, USA).UGT assays were incubated at 37 °C for 6 h in a final volume of 100 µL of assay buffer containing 20 µg of proteins, 0.5 mM DTT, 10 mM MgCl 2 , 50 mM Tris-HCl (pH 7.5), 20 µg/mL alamethicin, 2 mM UDP-GlcA and 0.5 µg/mL leupeptin with the indicated substrate.Samples were then stopped by adding 100 µL cold methanol, centrifuged at 16,000×g for 10 min and supernatants analysed by mass spectrometry.The following negative controls were included for each analysis: reaction assays without substrate, without the microsomal fraction and without substrate and microsomal fraction. ", "section_name": "Drug conjugation assays", "section_num": null }, { "section_content": "The establishment of these analytical methods required the optimisation of chromatographic conditions, production of glucuronide standards using pooled liver microsomes and the use of deuterated molecules as internal standards (Clearsynth, Mississauga, ON, Canada).Drugs and their G metabolites were measured on an API 6500 mass spectrometer (Sciex, Concord, ON, Canada), operated in multiple reactions monitoring mode and equipped with a turbo ion-spray source.Electrospray ionisation was performed in the positive ion mode.The chromatographic system consisted of a Nexera (Shimadzu Scientific instruments, Inc, Columbia, MD, USA).For fludarabine-G1 and G2, the chromatographic separation was achieved with an ACE Phenyl 3.0 µm packing material, 100 × 4.6 mm (Canadian Life Science, Peterborough, ON, Canada).The mobile phases were solvent A: water with 0.1% formic acid (v/v) and solvent B: methanol, at a flow rate of 0.9 ml/min.Fludarabine-G1 and G2 were eluted using the following programme: 0-2.0 min, isocratic 20% B; 2.0-2.1 min, linear gradient 20-65% B; 2.1-4.0 min, isocratic 65 % B; 4.0-4.1 min, linear gradient 65-90% B; 4.1-5.2min, isocratic 90 % B; 5.2-5.3min, linear gradient 90-20 % B; 5.3-8.5 min, isocratic 20% B. For cerdulatinib-G, chlorambucil dechlorinated metabolites G1 and G2, venetoclax-G, and the bendamustine derivative HP2-G, the chromatographic separation was achieved with a Gemini C18 3.0 µm packing material, 100 × 4.6 mm (Phenomenex, Torrance, CA, USA).The mobile phases were solvent A: 1 mM ammonium formate in water and solvent B: 1 mM ammonium formate in methanol at a flow rate of 0.9 ml/min.Cerdulatinib-G and chlorambucil dechlorinated metabolites G1 and G2 were eluted using the following programme: 0-5.0 min, linear gradient 10-90% B; 5.0-5.2min, isocratic 90% B; 5.2-5.3min, linear gradient 90-10% B; 5.3-8.3min, isocratic 10 % B. Venetoclax-G was eluted using the following programme: 0-2.0 min, linear gradient 10-90% B; 2.0-6 min, isocratic 90% B; 6.0-6.1 min, linear gradient 90-10% B; 6.1-9.2 min, isocratic 10% B. The bendamustine metabolite HP2-G, was eluted using the following programme: 0-5.0 min, linear gradient 20-90% B; 5.0-5.2min, isocratic 90% B; 5.2-5.3min, linear gradient 90-20% B; 5.3-8.3min, isocratic 20% B. For idelalisib-G1 and G2, the chromatographic separation was achieved with a Gemini C18 3.0 µm, 100 × 4.6 mm, using solvent A: 2 mM ammonium formate in water and solvent B: 2 mM ammonium formate in methanol at a flow rate of 0.9 ml/min.Idelalisib-G1 and G2 were eluted using the following programme: 0-3.0 min, isocratic 60 % B; 3.0-3.1 min, linear gradient 60-90% B; 3.1-4.0min, isocratic 90% B; 4.0-4.1 min, linear gradient 90-60% B; 4.1-7.0min, isocratic 60% B. For ibrutinib-G1 and G2, acalabrutinib-G and duvelisib-G, the chromatographic separation was achieved with an ACE Phenyl 3.0 µm, 100 × 4.6 mm, using a mobile phase of 75%, 75% and 65% methanol, respectively, eluted with 1 mM ammonium formate in water in isocratic mode at a flow rate of 0.9 ml/min.The systems were controlled through Analyst Software, version 1.6.1 from AB Sciex. ", "section_name": "Mass spectrometry-based analysis of drug conjugation", "section_num": null }, { "section_content": "Cells were plated at 1 × 10 4 cells/well (MEC1) or at 5 × 10 4 cells/ well (JVM2) in 96-well U-bottom tissue culture plates (BD Bioscience, Mississauga, ON, Canada).Drugs were added at time of plating at concentrations ranging from 1 nM to 100 µM (7-9 concentrations per drug), depending on the drug and based on cell viability.MTS cell viability assays (Aqueous One assay, Promega, Madison, WI, USA) were conducted 72 h after treatment initiation according to the manufacturer's instructions.Absorbance was read on a TECAN infinite M1000 plate reader (Tecan Group Ltd., Männedorf, Zurich, Switzerland) at 495 nm.Control cells were treated with corresponding vehicle concentration.To determine half maximal inhibitory concentrations (IC 50 ), MTS readout of control samples treated with vehicle only was set to 100% cell viability, and relative cell viability of drug-treated cells was determined by dividing MTS values of the treated samples by the control.IC 50 were calculated by fitting variable slope non-linear curves to normalised response data from drug treatments using GraphPad Prism v5 (GraphPad Software Inc., La Jolla, CA, USA).Assays were replicated at least three times in triplicates. Cytotoxicity of drug treatments was determined by labelling cells with AlexaFluor 647-conjugated Annexin V (Life Technologies Inc., CA, USA) and propidium iodide (Sigma).Cells plated in 96well plates at 2 × 10 4 cells/well (MEC1) or 3 × 10 4 cells/well (JVM2) were exposed to varying drug concentrations as above for 72 h.Cells were washed, labelled for 12 min with Annexin V (1:200) and PI (2 µg/ml) in Annexin V binding buffer, then immediately analysed by flow cytometry on a FACSCelesta equipped with a high throughput sampler (BD Bioscience).Annexin V labelling assays were replicated at least twice in duplicates. ", "section_name": "Drug sensitivity assays", "section_num": null }, { "section_content": "Total RNA was extracted from MEC1, EHEB and JVM2 cells treated with fludarabine, ibrutinib or idelalisib using RNeasy plus mini spin kits (Qiagen, Toronto, ON, Canada).cDNA was generated using SuperScript IV reverse polymerase (Thermo Fisher Scientific, Waltham, MA, USA).UGT genes were measured by qPCR analysis of 10 ng of cDNA with Power SYBR green master mix (Thermo Fisher Scientific).For measures of other genes including housekeeping genes from normalisation, total RNA was DNase I-treated and purified using the RNeasy MinElute Cleanup kit (Qiagen) per manufacturer's instructions and as described previously. 22RNA sequencing experiments were performed on MEC1, EHEB and JVM2 cells treated with fludarabine (10 µM and 50 µM), ibrutinib (1 µM and 5 µM) and idelalisib (5 µM and 10 µM) for 48 (MEC1), 72 (JVM2) or 96 (EHEB) hours.Sequencing data was quality trimmed using Trimmomatic v0.36 and aligned to GRCh38 using HISAT2 v2.1. 24Transcriptome assembly and generation of read-count matrices were performed using Ensembl GRCh38 transcriptome annotation with StringTie v1.3.4. 25 Differential gene expression analysis was done with the edgeR package for R v3.5.1.Isoform quantification was done using kallisto v0.44.0 with a custom GTF formatted annotation containing alternative UGT transcript information. 26Sequencing data are available from the Gene Expression Omnibus (GEO) accession number GSE135030. Public expression data from 291 CLL patients was downloaded from the International Cancer Genome Consortium (ICGC) with the project code CLLE-ES. 27Public microarray data from PBMCs of untreated CLL patients and CD19 + -sorted B-cells from patients treated with fludarabine-containing regimens were obtained from the Gene Expression Omnibus with the accession numbers GSE13159 28 and GSE15490, 23 respectively.In the latter study, responders included complete response or partial response, and non-responders comprised stable disease or progressive disease.Long-read sequencing data was downloaded from the Sequence Read Archive (SRA) using the accession number SRP036136 and aligned with HISAT before visualisation.Identification of possible upstream regulators was carried out using iRegulon 29 for Cytoscape v3.2.1.ENCODE ChIP-seq data from samples GM12891, GM12878 and GM19099 was analysed for RELA binding within a 10 kb window centred on the TSS of genes co-expressed with UGT2B17 by bootstrapping.Differential expression analysis for RNA-seq data was done using the edgeR v3.22.5, while microarray data was analysed with limma v3.36.5 and affy v1.58.Annotation-Hub v2.12.1, Iranges v2.14.12 and GenomicRanges v1.32.7 were used for analysis of ChIP-seq data.Clustering and functional enrichment analyses were performed using clusterProfiler v3.8.1, coseq v1.4.0,[32][33][34][35][36][37] Luciferase assays The sequence corresponding to 2.5 kbp of UGT2B17_n2 promoter (including exon 1c) was PCR amplified from LNCaP cell line genomic DNA using Phusion DNA Polymerase (NEB, Whitby, ON, Canada) as recommended, with the following primers (annealing at 63 °C): forward 5′-CTAGCAGACGCGTGAGATCCTAGTAGGAGGTT TTGGC-3′ and reverse 5′-CTAGCAGCTCGAGCAAGTTCCAGATGTC CAGACTC-3′.The PCR fragment was then digested with MluI and XhoI restriction enzymes and inserted in pGL3 basic vector (Promega) digested with the same enzymes, using the Rapid DNA Ligation Kit (Roche).Construct was verified by Sanger sequencing.MEC1 cells were co-transfected with 9.5 µg pGL3 constructs and 0.5 µg pRL-null basic renilla (Promega) using the Neon Transfection System (Thermo Fisher Scientific).Cells were then harvested, lysed and assessed for luciferase activity using the dual-luciferase reporter assay kit (Promega), as per manufacturer's instructions.Luciferase activity was calculated as the ratio of firefly luciferase to renilla activity, relative to the pGL3 control.Assays were replicated four times in triplicates. ", "section_name": "Gene expression analyses", "section_num": null }, { "section_content": "Results from glucuronidation assays represent a minimum of two independent experiments.All other results represent at least three independent experiments.Statistics were calculated using Graph-Pad Prism v5 (GraphPad Software Inc., La Jolla, CA, USA) or R v3.5.1.P-values were calculated using Student's T-test unless otherwise indicated.Differences in gene expression were considered significant if adjusted P-values were inferior to 0.05. ", "section_name": "Statistical analyses", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "In 20 CLL patients exposed to fludarabine-based regimens, 23 microarray data analysis of RNA from CD19 + -sorted B-cells (GSE15490) showed that UGT2B17 was induced shortly after treatment initiation in 6 of 11 non-responders whereas it was induced in only 2 responders out of 9 (P = 0.0007) (Fig. 1a).Patients showing an induced expression of UGT2B17 after treatment also displayed a 7.5-fold higher basal expression of UGT2B17 relative to those showing no induction (P = 0.0006), with all cases showing low expression of all other UGT isoforms.This was sustained by the analysis of RNA sequencing data from 291 CLL cases indicating that UGT2B17 predominates in leukaemic cells (Fig. 1b).Cells of MEC1 and JVM2 models overexpressing UGT2B17 were more resistant than cells with low expression, with half maximal inhibitory concentration (IC 50 ) values higher by 1.5 to 4.3-fold (P < 0.013) for fludarabine and ibrutinib (MEC1 and JVM2) as well as idelalisib, chlorambucil and venetoclax (MEC1) established by MTS assays (Table 1).Similar results were obtained using AnnexinV/PI labelling (data not shown).At clinically relevant concentrations, fludarabine, ibrutinib and idelalisib displayed no cytotoxicity in either cell lines, determined by Annexin V staining (Supplementary Fig. S1). ", "section_name": "UGT2B17 expression in B-cells is associated with reduced sensitivity to anti-leukaemic drugs and is induced by short-term drug treatment in patients", "section_num": null }, { "section_content": "As observed in CLL patients (Fig. 1a), fludarabine significantly induced UGT2B17 expression in MEC1, JVM2, and EHEB cellular models (Fig. 2a-c).UGT2B17 was also induced by ibrutinib and idelalisib (by 1.2 to 13.6-fold; P < 0.01) and was associated with elevated enzyme activity for DHT glucuronidation, a substrate for the UGT2B17 enzyme (1.9 to 10.5-fold; P < 0.01) (Fig. 2d).The low expression of UGT1A was also perturbed by each drug treatment (Fig. 2a-c).Consistent with these changes, glucuronidation activity was induced as demonstrated by increased levels of conjugate of the substrate oestradiol (E 2 ) (Fig. 2d).In the same line, fludarabine, ibrutinib and idelalisib increased fludarabine glucuronidation (Fig. 2e).As observed in CLL patients (Fig. 1b) comparative analysis of relative UGT expression levels further indicated a predominance of UGT2B17 (Fig. 2f).A more detailed assessment of the isoforms expressed identified UGT1A6 as the most abundant UGT1A isoform (Supplementary Fig. S2). Anti-leukaemic drugs are conjugated to GlcA in CLL-treated patients Given the higher and induced expression of UGT2B17 in patients not responding to fludarabine-containing treatment regimen, we explored whether UGT2B17 may be involved in the glucuronidation of the drug itself.We also examined the conjugation of additional agents relevant to CLL.Our observations in CLL patients treated with fludarabine raised the possibility that UGT2B17 may be involved in 23 a UGT2B17 is preferentially induced in CLL patients not responding to fludarabinecontaining regimen.Expression of UGT2B17 was determined in CD19 + -sorted cells of CLL patients before and three days after the first cycle of fludarabine treatment.Patients received standard doses of fludarabine and cyclophosphamide (FC) or FC with rituximab (FCR).Clinical response was assessed three months after treatment initiation. 23Responders displayed partial or complete remission, non-responders had stable or progressive disease.Expression of UGT2B17 is significantly enhanced (> 15 %; P = 0.0007) three days after treatment in the subset of patients not responding to fludarabine-containing regimen.CLL patients were dichotomised on the basis of UGT2B17 induction.Prior to initiation of treatment with a fludarabine-containing regimen, average levels of UGT2B17 were 4.1 and 7.0 log 2 -units (P = 0.0006, after normalisation by robust multi-array average) in patients exhibiting no induction and induction of UGT2B17 expression, respectively.Clinical characteristics of CLL patients are provided in Supplementary Table S6.b UGT2B17 predominates in leukaemic cells.Relative expression levels of UGT isoforms in a cohort of 291 CLL patients from the International Cancer Genomic Consortium (ICGC) demonstrate that UGT2B17 is the main UGT expressed in leukaemic cells.The proportion of patients expressing UGT2B17 and other UGTs is illustrated.Other UGTs include nine UGT1A (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9 and 1A10) and six UGT2B (2B4, 2B7, 2B10, 2B11, 2B15 and 2B28). the glucuronidation of the drug itself, and potentially also of B-cell receptor inhibitors ibrutinib and idelalisib, which contain functional hydroxyl and amino groups susceptible for conjugation into inactive glucuronides (G).Glucuronidation assays using pooled human liver microsomes enriched in UGT enzymes led to the formation of polar G derivatives.This was confirmed by their fragmentation patterns assessed by mass spectrometry (MS), namely a loss of the GlcA moiety corresponding to a m/z shift of 176 Da (Fig. 3a).Fludarabine was conjugated into two glucuronides G1 and G2, named according to their chromatographic resolution (Fig. 3a).Additional confirmation was achieved by the disappearance of G1 and G2 upon βglucuronidase hydrolysis (Supplementary Figure S3).Similarly, ibrutinib and idelalisib each led to two glucuronidated products (Fig. 3a). A second set of experiments identified the UGT enzyme(s) involved, based on UGT expressed in metabolic liver, kidney and intestine tissues, and individual recombinant UGT1A (n = 8) and UGT2B (n = 6) enzymes, using quantitative MS methods (Fig. 3b).UGT2B17 and UGT1A4 were the main conjugating enzymes for fludarabine glucuronidation (Fig. 3b), with a preferred formation of G2 over G1.This observation mirrored activity in livers, which express these UGTs.The confirmation of fludarabine-G formation in CLL patients was established in plasma of two cases collected in the first week after fludarabine treatment (Fig. 3c).Both fludarabine-G1 and G2 were measured in the first patient (16.4 and 11.7 pg/mL), whereas G2 (24.1 pg/mL) was detected in the second patient.For ibrutinib and idelalisib, G1 and G2 were formed only in the presence of UGT1A4 and livers, with similar kinetic parameters (Fig. 3b, Supplementary Table S1), supporting the implication of this sole enzyme.Their formation was further abolished by nearly 90% in the presence of a specific UGT1A4 inhibitor, hecogenin (Supplementary Table S1).The formation of ibrutinib-G was then established in serial serum samples from 15 CLL patients undergoing ibrutinib treatment collected at baseline (T 0 ), 3 weeks to 2 months (T 1 ), and between 4 and 9 months (T 2 ) after treatment initiation (Fig. 3d, Supplementary Tables S2 andS3).A predominance of G2 over G1 (ratio of 6.5) was noted, similar to what was observed in livers and UGT1A4.The formation of ibrutinib-G represented on average 24 % of the parent drug and was strongly correlated to levels of ibrutinib (R 2 = 0.917, P < 0.001) but with a considerable patient-to-patient variability (CV = 190%).Lastly, we explored whether glucuronidation may be involved in the conjugation of additional anti-leukaemics and the potential involvement of UGT2B17.MS analysis confirmed the formation of at least one G product by livers following incubations with bendamustine, chlorambucil and targeted agents venetoclax, acalibrutinib, cerdulatinib and duvelisib (Fig. 4).The UGT2B17 and UGT1A4 enzymes were predominantly involved in their inactivation by glucuronidation (Fig. 4). ", "section_name": "UGT2B17 expression in B-cell models is induced by short-term drug treatment", "section_num": null }, { "section_content": "To gain insights into the cellular pathways associated with high and inducible UGT2B17 expression, we initially established a transcriptional signature associated with elevated UGT2B17 expression in 448 untreated CLL samples (Fig. 5a).This signature was then examined in cell models expressing high levels of UGT2B17 as well as in drug-treated cells in which UGT2B17 expression was induced.First, clustering revealed transcriptomic changes associated with elevated UGT2B17 expression in untreated CLL patients that resembled those found in overexpression models (Fig. 5b).K-means clustering of gene expression data designated two clusters with globally up-regulated (cluster 2) or down-regulated (cluster 3) gene expression across all samples, suggesting that these clusters encompass changes connected to UGT2B17 levels rather than those produced by drugs.Cluster 2 contained the UGT2B17 gene whereas several genes of the AMP-activated protein kinase (AMPK) signalling pathway were significantly enriched in cluster 3 (Supplementary Table S4), and further validated by quantitative PCR (Fig. 5b,c). A second series of analysis focused on drug-related signatures.We observed that kinase inhibitor-treated cells clustered together whereas fludarabine-treated cells had a distinct expression profile, likely owing to different mechanisms of action (Fig. 5b).An upstream analysis for causal interpretation of the expression changes associated with UGT2B17 exposed an enrichment of nuclear factor kappa B (NF-κB) binding targets (Supplementary Table S5).The analysis of NF-κB ChIP-seq data (GM12891, GM12878 and GM19099) derived from tumour necrosis factor α (TNF-α)-treated B-cells further confirmed NF-κB as a key regulatory 'hub point'. An analysis of the UGT2B17 transcriptomes of CLL patients and leukaemic cell models revealed that the enzyme is largely expressed from alternative transcripts rather than the canonical v1 transcript (Fig. 5d,e).Although these alternative transcripts, named UGT2B17_n2, n3 and n4, encode the UGT2B17 enzyme, they are comprised of additional alternative exons that extend the 5' untranslated region (Fig. 5e).Their expression was also detected by RT-PCR amplification of full-length transcripts from CLL patients and Sanger sequencing of amplicons.Long-read sequencing data using PacBio SMRT technology also confirmed the expression of alternative UGT2B17_n2 in the GM12891 lymphoid cell line.The functionality of the regulatory sequences upstream of the novel exon 1c (P3) and exon 1b (P2) were evidenced in luciferase assays (Fig. 5f).Compared to the low expression derived from the canonical UGT2B17 promoter P1 (1.3-fold), P2 and P3 enhanced luciferase gene expression by 2.5-and 16.3-fold, respectively, in MEC1 cells (Fig. 5f). ", "section_name": "Transcriptional changes associated with high UGT2B17 in CLL patients and cell models", "section_num": null }, { "section_content": "Understanding mechanisms that contribute to intrinsic and acquired resistance to therapy is key to finding useful predictive markers and innovative strategies to prevent or overcome treatment resistance in CLL.Previous reports identified UGT2B17 as a prognostic marker and a potential therapeutic target. 17,20,21,38elalisib-G (pmol/min/mg protein) UGT2B17 modifies drug response in chronic lymphocytic leukaemia EP Allain et al. ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "A potential impact of UGT2B17 expression on clinical outcome in treated patients emerges with observations of poor response in fludarabine-treated patients expressing high UGT2B17 levels whereas drug treatment further induces the UGT2B17-associated metabolic capacity of lymphoid cells according to our findings. Our observations further support a significant impact of the UGT metabolic pathway on the inactivation of most anti-cancer agents used in CLL, including commonly used treatments fludarabine, ibrutinib, bendamustine, chlorambucil and emerging targeted therapies idelalisib, venetoclax, acalabrutinib, cerdulatinib and duvelisib.This resulted in reduced sensitivity of cells to several anti-leukaemics associated with high UGT2B17 expression.Our .The metabolite of bendamustine HP2 was glucuronidated.The HP2 metabolite has hydroxyl groups in place of chlorine atoms in bendamustine.Chlorambucil undergoes a similar process, generating a dechlorinated metabolite subsequently conjugated.Venetoclax is an orally available, selective, small molecule inhibitor of BCL2 approved by the US Food and Drug Administration for the treatment of patients with CLL.Acalabrutinib is an orally available, irreversible Bruton's tyrosine kinase (BTK) inhibitor in development designed to be more selective than ibrutinib. 10Cerdulatinib (PRT062070) is an investigational oral, dual spleen tyrosine kinase (Syk), janus kinase (JAK1/3) and tyrosine kinase 2 (TYK2) inhibitor for the treatment of haematological malignancies and approved for the treatment of peripheral T-cell lymphoma. 9Duvelisib is an oral, dual small molecule inhibitor of phosphatidylinositol 3-kinase (PI3K) δ and γ, approved for the treatment of relapsed or refractory CLL. 7A second set of experiments identified the UGT enzyme(s) involved, revealing a predominant role for UGT2B17 and UGT1A4.S4. findings indicate that this may be caused, at least in part, by direct glucuronidation of the drug by the UGT2B17 enzyme namely for fludarabine but would also involve other mechanisms for ibrutinib and idelalisib not inactivated by UGT2B17.By catalysing the transfer of GlcA from the co-substrate UDP-GlcA, UGT2B17 inactivates and detoxifies its substrates.This is supported by the detection of fludarabine-glucuronides in circulation of CLL patients that recently initiated fludarabine-based treatment.The glucuronidation pathway inactivates other nucleotide analogues such as ribavirin and cytarabine through a glioma-associated oncogene homologue 1 (GLI1)-dependent mechanism involving the regulation of UGT1A protein stability in AML. 18,19This differs from our observations in untreated CLL patients, in which UGT2B17 expression predominates, and where high UGT2B17 expression was associated with shorter treatmentfree and overall survival and more patients requiring treatment. 17,20,21This shows that for a significant proportion of high-risk CLL patients, UGT2B17 is expressed in cancer cells prior to treatment initiation with the potential to affect primary response to first line treatment such as fludarabine and ibrutinib.Once fludarabine treatment is initiated, an induction of UGT2B17 expression was observed in B-cells of CLL patients not responding to fludarabine as well as in lymphoid cell models. 17,23reatment with targeted agents ibrutinib and idelalisib also resulted in a marked transcriptional up-regulation of UGT2B17, and high UGT2B17 expression was associated with reduced sensitivity to these drugs in B-cell models.While an influence of other drugrelated mechanisms, including transporters and drug metabolising enzymes is possible, CYP3A4, which is another key metabolising enzyme for these drugs, 39 was not perturbed in patients treated with fludarabine (not shown).It raises the possibility that therapeutic pressure induces UGT expression in B-cells.This rapid adaptation of neoplastic cells in the presence of a cytotoxic stressor and targeted therapies support that the UGT metabolic pathway is highly relevant in leukaemia, has the potential to affect drug response locally in malignant cells, and may be useful in predicting response to several CLL therapies.We also demonstrated that the glucuronidation pathway is involved in the conjugation of a number of additional anti-leukaemics (chlorambucil, bendamustine, venetoclax, acalabrutinib, cerdulatinib and duvelisib) and that UGT2B17 plays a role in the inactivation of chlorambucil and cerdulatinib.These two anti-leukaemic agents target different cellular pathways than fludarabine and have distinct modes of action.High UGT2B17 expression may thus lead to lower response to these drugs in CLL patients but this remains to be demonstrated. Crucial parts of the machinery governing UGT2B17 transcription remain poorly understood and especially in lymphoid cells.We provide the first evidence that UGT2B17 expression in B-cells is driven by a non-canonical UGT2B17 promoter and the use of an alternative noncoding exon 1c coupled to the common proteincoding region leading to the canonical UGT2B17 enzyme.According to our analysis, the gene expression signature associated with high UGT2B17 expression in CLL patients and cell models comprises a number of genes targeted by NF-κB.This promoter may thus be targeted by NF-κB that plays a central role in CLL.1][42][43][44] The mechanism underlying high UGT2B17 expression in B-cells remains to be fully explored. Our data also point to other mechanisms of altered drug sensitivity associated with high UGT2B17 expression.The inactivation of ibrutinib and idelalisib is largely dependent on UGT1A4 as the conjugation of other anti-leukaemics tested herein (chlorambucil, bendamustine, venetoclax, acalabrutinib, cerdulatinib and duvelisib).UGT1A4 is far less abundant than UGT2B17 in leukaemic B cells, and undetected at the mRNA level in most untreated CLL patients.Given that ibrutinib is mainly administered as an oral agent and subjected to first-pass hepatic metabolism, this drug is likely conjugated primarily in the liver expressing high levels of UGT1A4.In one of the lymphoid cell models, we showed drug-mediated induction of UGT1A4, but this finding remains to be demonstrated in CLL patients.Induction at the protein level also requires examination, given the recent report in drugresistant AML cells suggesting reduced mRNA expression but an enhanced UGT1A protein expression mediated by increased protein stability upon drug treatment. 18onsistent with its regulatory function of the levels of endogenous molecules, high UGT2B17 may deplete intracellular metabolites leading to aberrant cell signalling and dysregulated cell functions, 22 which could favour progression in untreated CLL cases and subsequent drug resistance in treated patients.In our transcriptomic analysis, AMPK signalling was negatively associated with high UGT2B17 expression in conditions of both induced and high basal UGT2B17 expression in cell models and CLL patients, potentially linking B-cell metabolism to the glucuronidation pathway and subsequent adverse clinical outcomes.AMPK is a major regulator balancing energy supply and ultimately protects cells from harmful stresses by the coordination of multiple metabolic pathways. 45The activation of the AMPK pathway has been shown to affect growth and apoptosis in CLL. 46As a stressresponse molecule mediating drug resistance through different mechanisms, AMPK is further involved in the metabolism reprogramming and induction of autophagy, also regulating the self-renewal ability of cancer stem cells. 47More recently, ibrutinib resistance was associated with a metabolic rewiring in CLL. 48ikewise, UGT proteins were shown to be part of complex protein networks.Their functional interaction with other metabolic proteins induced broad changes in cell metabolism and may contribute to tumorigenesis and drug response. 49,50he findings of this study have to be seen in light of some limitations, including the fact that drug cytotoxicity and treatment outcome in relation to UGT2B17 expression was investigated in cell models and a limited number of fludarabine-treated patients.However, IC 50 values of tumour cells, namely for ibrutinib, were in the same range as those reported previously in B lymphoblastoid leukaemic cell lines and in primary cells from patients, [51][52][53] supporting the relevance of our initial observations.The impact of UGT2B17 expression on response to drug in vivo and ex vivo, as well as the influence of the microenvironment on UGT2B17related responses, remains to be examined in more details. In summary, we unveiled a biochemical underpinning of reduced drug sensitivity related to the UGT2B17 metabolic pathway and drug inactivation.The evidence provided should prove useful for understanding and potentially overcoming drug refractoriness.The impact of glucuronidation in the inactivation of Fig. 5 Ascertaining pathways affected by high UGT2B17 expression in B cells and upstream regulators.a Schematic overview of the analysis pipeline.K-means clustering, enrichment and co-expression analyses served to identify pathways associated with high UGT2B17 expression and upstream transcriptional regulators.Detailed analysis of UGT2B17 transcriptome by RNA-seq revealed expression of noncanonical transcripts and alternate regulation.b A transcriptional signature associated with elevated UGT2B17 expression in PBMCs from 448 untreated CLL patients (GSE13159) 28 enabled clustering and identification of pathways altered by elevated UGT2B17 expression.The top 1000 most statistically significant genes were selected as features for clustering RNA-seq samples with log2 fold-change values using the K-means method.Cluster 3 contained genes which were globally down-regulated across all experimental conditions and was enriched with genes belonging to the AMPK signalling pathway.c RT-qPCR validation of down-regulated AMPK pathway-related genes in cell models.d UGT2B17 is predominantly expressed from an alternative UGT2B17_n2 transcript in lymphoid cell models and in CLL patients (n = 6; GSE99724) evaluated by RNA sequencing.e Schematic overview of the UGT2B17 gene and main coding transcripts in leukaemic cells.Only exons included in main leukaemic UGT2B17 transcripts are shown for sake of clarity.The alternative UGT2B17_n2-n4 transcripts include the supplementary exon 1c or 1b previously reported, which extend the 5′ untranslated sequence relative to the canonical UGT2B17_v1 transcript. 26UGT2B17_n2 and n4 transcripts were validated by RT-PCR in cells of three CLL patients, and in MEC1 and JVM2.These transcripts encode a functional UGT2B17 enzyme.f Luciferase reporter gene expression assays were performed in MEC1 cells.Cells were transfected with pGL3 vectors containing either the canonical promoter of UGT2B17_v1 (P1), or the alternative promoters P2 or P3 upstream of each novel exons 1b or 1c, respectively.Experiments were conducted four times in triplicates. a number of anti-leukaemic drugs is underestimated since we established that most agents are subjected to this metabolic process, including ibrutinib, idelalisib, venetoclax and duvelisib as well as other small molecules under development such as acalibrutinib and cerdulatinib.This may well apply to a number of other cancer therapeutics given the recent report that GLI1inducible glucuronidation imparts resistance to a broad spectrum of compounds including FDA-approved drugs such as methotrexate. 19Our observations warrant additional studies to appreciate the prevalence and the clinical implications of high UGT2B17 expression on outcomes of leukaemia patients. ", "section_name": "MS Fragmentation Chromatograms", "section_num": null } ]	[ { "section_content": "We would like to thank Isabelle Laverdière and Dominic Bastien for their help with the FACS analysis of leukaemic cells, and Etienne Audet-Walsh for his helpful advices with expression analyses. ", "section_name": "ACKNOWLEDGEMENTS", "section_num": null }, { "section_content": "The datasets generated during the current study are available in the Gene Expression Omnibus repository with the accession number GSE135030. ", "section_name": "Data availability", "section_num": null }, { "section_content": "Study concept: C.G. Performed experiments: E.P.A., S.T., J.V., V.B., P.C., L.V., A.L., V.T. Analysed data: E.P.A., M.R., S.T., J.V., V.B., P.C., L.V., V.T., C.J.B., E.L., C.G. Patient recruitment and clinical data: K.V., T.L., M.S., S.S., D.D., R.H., U.J., P.S. Drafting of the paper: E.P.A., M.R., C.G. Critical revision of the paper for intellectual content: all authors.Obtaining funding: E.L. and C.G. Ethics approval and consent to participate The study was carried out in accordance with the Declaration of Helsinki.Patients provided informed consent and the study was approved by the local ethical research committees of the Medical University of Vienna (Ethics vote 1499/2015) and the CHU de Québec (A14-10-1205). Consent to publish Not applicable. The authors declare no competing interests.Supplementary information is available for this paper at https://doi.org/10.1038/s41416-020-0887-6. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "Study concept: C.G. Performed experiments: E.P.A., S.T., J.V., V.B., P.C., L.V., A.L., V.T. Analysed data: E.P.A., M.R., S.T., J.V., V.B., P.C., L.V., V.T., C.J.B., E.L., C.G. Patient recruitment and clinical data: K.V., T.L., M.S., S.S., D.D., R.H., U.J., P.S. Drafting of the paper: E.P.A., M.R., C.G. Critical revision of the paper for intellectual content: all authors.Obtaining funding: E.L. and C.G. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "Ethics approval and consent to participate The study was carried out in accordance with the Declaration of Helsinki.Patients provided informed consent and the study was approved by the local ethical research committees of the Medical University of Vienna (Ethics vote 1499/2015) and the CHU de Québec (A14-10-1205). Consent to publish Not applicable. ", "section_name": "ADDITIONAL INFORMATION", "section_num": null }, { "section_content": "The authors declare no competing interests.Supplementary information is available for this paper at https://doi.org/10.1038/s41416-020-0887-6. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Funding information", "section_num": null } ]
10.1186/1479-7364-7-10	Extrapolating the effect of deleterious nsSNPs in the binding adaptability of flavopiridol with CDK7 protein: a molecular dynamics approach	Recent reports suggest the role of nonsynonymous single nucleotide polymorphisms (nsSNPs) in cyclin-dependent kinase 7 (CDK7) gene associated with defect in the DNA repair mechanism that may contribute to cancer risk. Among the various inhibitors developed so far, flavopiridol proved to be a potential antitumor drug in the phase-III clinical trial for chronic lymphocytic leukemia. Here, we described a theoretical assessment for the discovery of new drugs or drug targets in CDK7 protein owing to the changes caused by deleterious nsSNPs.Three nsSNPs (I63R, H135R, and T285M) were predicted to have functional impact on protein function by SIFT, PolyPhen2, I-Mutant3, PANTHER, SNPs&GO, PhD-SNP, and screening for non-acceptable polymorphisms (SNAP). Furthermore, we analyzed the native and proposed mutant models in atomic level 10 ns simulation using the molecular dynamics (MD) approach. Finally, with the aid of Autodock 4.0 and PatchDock, we analyzed the binding efficacy of flavopiridol with CDK7 protein with respect to the deleterious mutations.By comparing the results of all seven prediction tools, three nsSNPs (I63R, H135R, and T285M) were predicted to have functional impact on the protein function. The results of protein stability analysis inferred that I63R and H135R exhibited less deviation in root mean square deviation in comparison with the native and T285M protein. The flexibility of all the three mutant models of CDK7 protein is diverse in comparison with the native protein. Following to that, docking study revealed the change in the active site residues and decrease in the binding affinity of flavopiridol with mutant proteins.This theoretical approach is entirely based on computational methods, which has the ability to identify the disease-related SNPs in complex disorders by contrasting their costs and capabilities with those of the experimental methods. The identification of disease related SNPs by computational methods has the potential to create personalized tools for the diagnosis, prognosis, and treatment of diseases.Cell cycle regulatory protein, CDK7, is linked with DNA repair mechanism which can contribute to cancer risk. The main aim of this study is to extrapolate the relationship between the nsSNPs and their effects in drug-binding capability. In this work, we propose a new methodology which (1) efficiently identified the deleterious nsSNPs that tend to have functional effect on protein function upon mutation by computational tools, (2) analyze d the native protein and proposed mutant models in atomic level using MD approach, and (3) investigated the protein-ligand interactions to analyze the binding ability by docking analysis. This theoretical approach is entirely based on computational methods, which has the ability to identify the disease-related SNPs in complex disorders by contrasting their costs and capabilities with those of the experimental methods. Overall, this approach has the potential to create personalized tools for the diagnosis, prognosis, and treatment of diseases.	[ { "section_content": "Cyclin-dependent kinase 7 (Cdk7), a regulatory enzyme for the initiation of cell cycle progression, was initially identified from a search for cDNA encoding protein kinase(s) related to Cdk1 [1].For activation of Cdk1, Cdk2, Cdk4, and Cdk6, the catalytic subunit of the Cdk-activating kinase requires the association of Cdk7 with a regulatory subunit, cyclin H and the phosphorylation of a conserved threonine residue at position 170 within its own T loop [2,3].Subsequently, both CDK7 and the partner cyclin H were found to be associated with the general transcription factor TFIIH, suggesting additional roles of CDK7 in transcription.Given that CDK7 activates the main CDKs at different cell cycle transitions, it is possible to assume that the over expression of CDK7 also contributes to breast cancer cell proliferation [4].In addition, CDK7 plays a vital role in human DNA repair mechanism (in NER pathway).Evidences support the hypothesis that mutations are early events in carcinogenesis, so the defects in DNA repair probably represent a high risk factor for many types of cancer [5,6].Consistent with these actions, CDK7 was treated as a potent therapeutic target to inhibit the activity of cell cycle in cancerous cells.Currently, in phase-III trials for chronic lymphocytic leukemia, flavopiridol a potential antitumor drug has shown better inhibitory effect towards CDK7 [7].It is known that flavopiridol decreases transcription by inhibiting CDK7 [8], which is responsible for the phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II, an activity essential for both transcriptional initiation and elongation [9,10].Analyzing the human genetic variation promises to have a significant impact on the ability to understand the basis of individual variation in response to therapeutics.As we are entering the age of \"personalized genomics\", it is expected that the knowledge of human genetic variations could provide a basis for understanding the differences in susceptibility to diseases and designing individualized therapeutic treatments [11,12].It was estimated that 90% of human genetic variations were caused by single nucleotide polymorphisms (SNPs) [12].For example, changes in amino acids of proteins, such as the nonsynonymous single nucleotide polymorphisms (nsSNPs) in the gene coding regions could account for nearly half of the known genetic variations linked to human inherited diseases [13].The nsSNP might change the physicochemical property of a wild-type amino acid that affects the protein stability and dynamics and disrupts the interacting interface, protein-small molecule, and proteinprotein interaction [14][15][16][17].Taken together, single mutation may affect binding ability of the inhibitory molecule.Recent progress in high throughput human genome research has provided a wealth of information detailing tens of millions of human genetic variations between individuals, including SNPs [11,18].Numerous efforts have been carried out to illustrate how nsSNPs produce deleterious effects on the stability and function of a protein [19][20][21][22][23].Given the large number of SNPs, a detailed experimental study on the effect of mutation in biological function is a daunting task.An effective alternative is the use of in silico methods.These approaches were based on the biochemical severity of the amino acid substitution, as well as the protein sequence and/or structural information, which can provide a more feasible method for phenotype prediction. Recently, more sophisticated in silico algorithms were developed to predict the impact of amino-acid substitutions on protein structure and function.Some of the variation tolerance methods follow a similar procedure, in which a missense variant is first labeled with properties, related to the damage it may cause to the protein structure or function [24].However, in other methods, predictions are based on the difference in the free energy of unfolding (DDG) between a native-type and mutant protein.The methods that use energy functions can be subdivided into physical, statistical, and the empirical potential approaches [25].The ultimate goal of all these approaches is to determine the deleterious nsSNPs from the neutral ones.In general, in silico methods can provide a feasible and the highthroughput way to determine the impact of large numbers of nsSNPs on protein function.To understand the atomistic level changes and the dynamic behavior of the molecule with respect to the potential mutations, we conducted molecular dynamics (MD) simulations analysis.MD simulations can help us understand the effects of mutation on protein structure, which allow exploring how one amino acid substitution can create a ripple effect throughout the protein structure.Offman et al. found a strong correlation between MD analysis and the experimental work on the molecular basis of the most common protein upon N370S mutation in causing Gaucher's disease [26,27].Thus, we assume that MD simulation analysis might provide more reliable structural information upon CDK7 mutations. Although deleterious nsSNPs of CDK7 gene have received considerable attention from experimental biologists, the functional consequence of most of the nsSNPs in CDK7 at the structural level is still unknown.The main goal of this in silico analysis is to determine the most deleterious variants in CDK7 gene.In this context, publicly available in silico tools such as Sorting Intolerant From Tolerant (SIFT; J. Craig Venter Institute, Rockville, USA) [28], Polymorphism Phenotyping (PolyPhen) version 2 [29], PANTHER [30], I-Mutant3 [31], SNPs&GO (Bologna Biocomputing Group, Bologna, Spain) [32], predictor of human deleterious single nucleotide polymorphisms (PhD-SNP; Bologna Biocomputing Group) [33], and screening for non-acceptable polymorphisms (SNAP; Bologna Biocomputing Group) [34] were used to analyze the nsSNPs in CDK7 gene.As a next step, we subjected MD simulation study in the native and mutant models of CDK7 proteins using GROMACS 4.5.3package [35,36].MD simulations will reveal the level of structural conformations changes with respect to the incorporation of deleterious mutations in CDK7 protein.Finally, the binding capability of CDK7 inhibitor, flavopiridol, was analyzed with respect to the structural mutations.Docking study was carried out with the help of AutoDock4 (The Scripps Research Institute, La Jolla, USA) and PatchDock [37][38][39].The proposed protocol is represented schematically in Figure 1. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Dataset for the evaluation of potential nsSNPs in CDK7 gene was retrieved from dbSNP [40] and SwissProt [41] database.We selected 14 nsSNPs for further consideration, and their associated biomedical informations were retrieved from OMIM (Johns Hopkins University, Baltimore, USA) [42], PubMed, and Swiss-Prot database.Related experimental data about the CDK7 protein and Protein Data Bank (PDB) structural information with PDB ID 1UA2 [43] were obtained from Swiss-Prot database and PDB, [44] respectively.The ligand molecule, flavopiridol, was obtained from Drug Bank database [45]. ", "section_name": "Dataset", "section_num": null }, { "section_content": "Identifying the deleterious nsSNPs has become possible with the aid of improved in silico algorithms.Here, we analyzed 14 nsSNPs of CDK7 gene with seven different in Figure 1 Outline of proposed protocol for nsSNPs analysis.This protocol explains the different steps followed in nsSNP analysis via experimental (grey color) and computational methods.Box displayed in orange color indicates the effectiveness of computational over experimental methods. silico tools specifically SIFT, PolyPhen2, I-Mutant3, PAN-THER, SNPs&GO, PhD-SNP, and SNAP to determine the protein structural and functional significance.Table 1 displays the distribution of the deleterious and neutral variations of CDK7 gene with the corresponding amino acid substitution.SIFT makes inferences from sequence similarity using mathematical operations.SIFT constructs a multiple sequence alignment (MSA) and considers the position of the missense variants.Based on the amino acids appearing at each position in the MSA, SIFT calculates the probability and classifies a missense variant 'tolerated'.SIFT can be applied not only to naturally occurring nsSNPs but also to identify artificial missense mutations.Among the 14 nsSNSPs analyzed by SIFT, six were identified as deleterious which obtained a SIFT score ≤0.05.PolyPhen2 utilizes a combination of sequence and structure-based attributes for the description of an amino acid substitution, and the effect of mutation is predicted by a native Bayesian classifier.The sequence-based features include position specific independent count (PSIC) scores, MSA properties, and position of mutation in relation to domain boundaries as defined by Pfam [46].The structure-derived features are solvent accessibility, changes in solvent accessibility for buried residues, and crystallographic B-factor.By PolyPhen2, nine nsSNPs were predicted as probably and possibly damaging, having the effect on protein structure and function of CDK7 protein; the remaining five were classified as benign which obtained score less than 0.15.In order to verify the prediction accuracy of SIFT scores, we used hidden Markov model (HMM)-based evolutionary approach PANTHER to verify the effect on protein function upon a single point mutation.Out of 14 nsSNPs, 7 were designated as deleterious with a score of ≤-3.In order to improve overall prediction accuracy, we used I-Mutant3, a support vector machine-based stability prediction tool.A score less than '0' means the mutation decreases the stability.The smaller the score, the more certain is the prediction.Conversely, a score more than '0' means mutation increases the protein stability.Among the 14 nsSNPs of the CDK7 gene, 13 nsSNPs showed negative DDG values, were considered to be less stable and deleterious.The remaining one nsSNP showed a positive DDG value and classified as non-deleterious.SNPs&GO is an support vector machines (SVM) classifier based on variation type and sequence environment information, sequence profiles taken from MSAs, predictions from the program PANTHER, and a function-based log-odd score describing information about protein function defined by Gene Ontology (GO) terms.SNPs&GO predicted four nsSNPs of CDK7 gene, which are related to a disease condition.SNAP is a neural network-based method that uses in silico derived protein information (e.g., secondary structure, conservation, solvent accessibility, etc.) in order to make predictions regarding functionality of the mutated proteins.The network takes protein sequences and lists of mutants as input, returning a score for each substitution.These scores can then be translated into binary predictions of effect (present/absent) and reliability indices.SNAP screened four nsSNPs of CDK7 gene as non-neutral, and it may cause phenotypic changes.PhD-SNP is a prediction method based on single sequence profile-based SVM, trained on Swiss-Prot variants.The single sequence SVM classifies the missense variant to be pathogenic or neutral, based on the nature of substitution and properties of the neighboring sequence environment.PhD-SNP classified four nsSNPs as deleterious.Comparing the results of all seven prediction tools, three nsSNPs at corresponding amino acid position I63R, H135R, and T285M with a highest SIFT tolerance index of 0.00 and PSIC score difference 1.0 were selected for structural analysis. ", "section_name": "Prediction of deleterious nsSNPs by in silico tools", "section_num": null }, { "section_content": "Structural information could play a vital role in unveiling the molecular mechanisms leading to a disease.Based on this, we proposed modeled structures for all the three mutants (I63R, H135R, and T285M) of CDK7 protein using PyMOL (Schrödinger, Bangalore, India) [47].Substitution of an amino acid may produce changes at the structural level.Changes in the secondary structure with respect to the substituted amino acid were analyzed in PDBsum (Cambridge, UK).Additional file 1: Figure S1 displays the secondary structural elements of the native and mutant models.The number of secondary structure elements such as beta sheets, beta hairpins, beta bulges, strands, helices, helix-helix interactions, beta turns, and gamma turns was calculated for both the native and mutant models (Table 2).It has to be noted that the observed numbers of secondary structural elements are equal in both native and mutant models except the turns.There was a slight increase in the number of beta turns in all the three mutant models.The native protein exhibited only 30, while mutants obtained 31 beta turns.Substitution of arginine in the modeled H135R protein leads to decrease in the number of gamma turns as six, whereas the native and remaining mutants I63R and T285M obtained seven gamma turns.Further, the surrounding amino acid residue changes were visualized from the point of mutational position.A residue change within 4A°surroundings was observed through PyMOL (Figure 2A-C).In addition, the number of cation-pi interacting residues for both the native and three mutants was calculated using Protein Interactions Calculator server [48].A cation-pi interaction plays a vital role in maintaining the protein structural stability and is recognized as an important non-covalent binding interaction in structural biology [49,50].Change in the secondary structural elements may bring about some changes in the cation-pi interacting residues in the mutant models.It has to be noted that the number of intramolecular cation-pi interactions in the native protein is seven.The substitution of deleterious amino acid increased the number of cation-pi interactions in the mutants I63R, H135R, and T285M as eight, nine, and eight, respectively (Additional file 2: Table S1).Cation-pi interacting residue distances and angles varied in mutant model showed the deleterious effects of substituted amino acid.Overall, the structural analysis results inferred that the three deleterious mutations had brought a drastic change in the CDK7 protein, and it could affect the protein function. ", "section_name": "Analysis of secondary structure and surrounding amino acid changes", "section_num": null }, { "section_content": "In vitro studies of flavopiridol showed inhibitory activity towards CDK7 protein and lead to programmed cell death in cancerous cells [51].Substitution of deleterious amino acid in CDK7 protein may affect the binding ability of CDK7 with flavopiridol.This has to be analyzed to improve the potentiality of the drug to inhibit CDK7 protein.Hence, we analyzed the binding ability of flavopiridol with native and mutant models of CDK7 protein using in silico docking tool, Autodock4 and PatchDock [39].Before entering into docking analysis, we evaluated the binding sites of native CDK7 protein.Flavopiridol binds at the ATP binding site of the native CDK7 protein and made contact with 12 amino acid residues.Twelve residues specifically GLY21, GLN22, PHE23, ALA24, VAL26, LYS41, PHE91, ASP97, ASN141, LEU144, ALA154, and SER161 were involved in proteinligand interaction.This information was in concordance with the study conducted by Carlson et al. [51] and Worland et al. [52].In their analysis, it was observed that flavopiridol directly inhibits CDK7 by competing for to the ATP binding site.In addition, these 12 residues were also involved in protein-ATP interaction observed by Lolli et al. [2] in their crystallography analysis.In the mutant models (I63R, H135R, and T285M), we observed the number of contact residues as six, seven, and six, respectively (Additional file 3: Table S2).Decrease in the number of residue contacts will definitely affect the complementarities between mutant protein and flavopiridol compound as shown in Figure 3A-D.Further, we observed the interaction of flavopiridol with native and mutant proteins by LIGPLOT (Additional file 4: Figure S2).Shape complementarity and non-covalent interactions were believed to drive protein-ligand interaction.Non-covalent bonds such as hydrogen bonds, van der Waals contacts and electrostatic forces are the dynamic forces involved in protein-ligand interactions.Calculating the interaction energies of noncovalent bonds is a key point in understanding the binding efficiency of a ligand molecule.The number of hydrogen bonds formed between protein and ligand, and van der Waals interacting energies and electrostatic interacting energies was computed using Autodock4.The binding energy and the non-covalent bond interaction energy between CDK7 protein (native and mutant) and flavopiridol molecule were calculated and shown in Table 3.In the native complex, the significant contribution of van der Waals and electrostatic energy was observed as -9.18 and -9.07 kcal/ mol, respectively.On the contrary, mutant models I63R, H135R, T285M interacting with flavopiridol showed an increase in van der Waals and electrostatics energies as -5.52, -5.53, and -5.57, and -0.65, -0.6, and -0.68 kcal/ mol, respectively.The total ligand receptor binding energy exhibited by the native, I63R, H135R, and T285M complexes were -8.27, -5.57, -5.53, and -5.65 kcal/mol, respectively.Lower binding energy of native complex indicates better interaction and good compatibility with the flavopiridol compound. In order to determine the consistency in docking analysis, in addition to Autodock4 analysis, we performed docking in PatchDock [39].Docking was performed between the drug flavopiridol with both the native type and mutant modeled structures of CDK7 protein to find out the binding efficiency in the form of PatchDock score and atomic contact energy (ACE) values (Table 4).In this analysis, we found that native type CDK7 protein obtained high PatchDock score and ACE as 5,956 and -0.25, respectively.But, all the three mutant structures (I63R, H135R, and T285M) obtained less PatchDock scores (4,148, 4,136, and 4,978) and high ACEs (8.98, 19.92, and 21.05).Notably, high PatchDock score and less ACE value were obtained in the native complex which is considered as a good docked complex than those of other three mutant complexes.This implies the concordances of computational algorithms in docking analysis and gives a 'theoretical quantitative' assessment on the binding efficiency of CDK7 mutant protein with flavopiridol. ", "section_name": "Docking analysis", "section_num": null }, { "section_content": "Molecular dynamic simulations studies were carried out to unravel the atomic level changes in the CDK7 protein with respect to the time scale.The overall protein stability changes upon mutation were evaluated by root mean square deviation (RMSD) values.We calculated the backbone RMSD for all the atoms from the initial structure, and this is considered to be a primary criterion to measure the convergence of the protein system concerned.The backbone RMSD was calculated for both the native and mutant models from the appropriate trajectory files (Figure 4).We observed a significant structural deviation in the mutant proteins I63R, H135R, and T285M when compared to native CDK7 protein structure.All the four structures attained a significant deviation at last 5 ns.The native and T285M mutant structure obtained a mean RMSD of approximately 0.35 nm in the last 5 ns, and mutant models I63R and H135R exhibited a deviation range from about 0.25 to 0.3 nm.This difference in the deviation range in mutant model explains the stability change and reflects the impact of substituted amino acid in the protein structure.In order to determine the structural flexibility of both the native and mutant models of CDK7 protein, we calculated the root mean square fluctuation (RMSF) values from the 10 ns simulation trajectory data.The RMSF values of native and mutant models are shown in Figure 5.In the entire 10 ns simulation period, native residues from approximately 25 to 150 nm showed a high fluctuation in comparison with I63R, H135R, and T285M mutant models.In the remaining residue range from around 150 to 260 nm, the mutant model I63R exhibited high fluctuation.Overall, RMSFs of all the mutant models were significantly deviated from the native structure in the entire simulation period.A change in the RMSFs specify the mode of flexibility changes in the mutant models and reflects the impact of deleterious amino acid substitution in CDK7 protein.Salt bridge distances of CDK7 protein in both native and mutant proteins were calculated from the 10 ns trajectory data and shown in Additional file 5: Figure S3.In a period of about 1,000, 5,000, and 7,000 ps, native protein obtained a low salt bridge distance of approximately 0.2 nm and maintained an average range distance around 0.45 nm throughout the 10-ns simulation period.Two mutant models (I63R and H135R) maintained a similar distance like the native protein.The mutant model T285M exhibited high range of salt bridge distance in the maximum simulation period when compared to the native, I63R, and H135R structures.From this analysis, we conclude that salt bridges are more stable in I63R and H135R mutant models when compared to the mutant model T285M. ", "section_name": "Molecular dynamics, structural stability, and flexibility analysis", "section_num": null }, { "section_content": "The central objective in molecular biology and population genetics is to identify and characterize the nsSNPs that are functionally related from those that are not.This understanding not only provides insight into cancer biology but also highlights the anticancer therapeutic targets and diagnostic markers.NsSNPs in coding region can lead to amino acid change.This can lead to alterations in protein function and account for susceptibility to disease and altered drug response.Identification of deleterious nsSNPs from tolerant nsSNPs is ideal for analyzing individual susceptibility to disease, understanding the pathogenesis of disease, identifying molecular targets for drug treatment, and conducting individualized pharmacotherapy.Several experimental studies were carried out to analyze the relationship between nsSNPs and drug response in cancer treatments.Chambers et al. [58] reported the involvement of nsSNPs in the modulation of protein structure and function.Another finding by Giovannetti et al. [59] demonstrated the role of nsSNPs in the DNA-repair protein to be the potential biomarkers of primary resistance to gemcitabine/cisplatin-based polychemotherapeutic agent in the treatment of pancreatic cancer.In another analysis, Wang and Moult [60] reported the role of nsSNPs in individuals by inducing or influencing the disease by affecting protein-protein interactions, protein expression, alternative splicing, stability, folding, and ligand binding or catalysis. These mounting studies on nsSNPs assert their role in better understanding the resultant phenotypic variations among individuals with an endeavor towards new drug design and development.The exponential increase in the number of SNPs makes the determination of biological significance of each nsSNP by wet laboratory experiments impossible.Alternatively, in silico programs and statistical methods may be used to predict the effects caused by mutations and elucidate the underlying biological mechanisms.However, in silico tools can be used to examine the potentially deleterious nsSNPs that might affect important drug targets before further investigation by wet laboratory techniques.Previously, our group also identified and analyzed the effects of deleterious nsSNPs in several proteins at structural and functional level and drug binding capability using various in silico tools [61][62][63].In this paper, we performed a systematic in silico analysis to determine the potential deleterious and functional nsSNPs in CDK7 protein along with molecular dynamics and docking study. To determine the possible effects of nsSNPs in CDK7 gene, we employed seven widely used in silico tools specifically SIFT, PloyPhen2, I-Mutant3, PANTHER, SNPs&GO, SNAP, and PhD-SNP.SIFT predicted 6 (42.85%) nsSNPs as deleterious, PolyPhen2 identified 9 (64.28%)substitutions which affect protein structure and function, I-Mutant3 identified 13 (92.85%)substitutions which affected the stability of protein, PAN-THER predicted 7 (50%) nsSNPs to be deleterious, SNPs&GO, SNAP, and PhD-SNP identified 4 (28.57%)nsSNPs related to the disease condition.The basis for the predicting impact of nsSNPs in these seven algorithms was different, and we would expect the outcomes to occur in some ways, dissimilar.However, the positive predictions that overlap all these seven in silico tools would provide high reliability to behave similarly.The difference in their predictions might be due to the difference in features utilized by the methods or the training dataset.Comparing the prediction of all the seven methods, three nsSNPs (I63R, H135R, and T285M) were identified as highly deleterious and selected for further structure and functional investigations.To gain insight knowledge on the protein structure and what kind of harmful modulation these mutations give rise, the CDK7 protein was analyzed by MD approach.In the 10 ns simulation trajectory, different parameters were applied to analyze the level of structural changes.Molecular stability and flexibility changes were observed by RMSD and RMSF analyses.Stability is a fundamental property affecting the bimolecular function, activity, and regulation.Protein stability analysis results inferred that the stabilities of I63R and H135R RMSD are less deviated than those of the native and T285M protein.High or less deviation implies increase or decrease in the stability of protein.Hence, we believe that reduction in the stability of I63R and H135R models could affect the CDK7 protein structure.From the fluctuation analysis, we observed a decrease in the flexibility for all the three mutant models in first half of the residues (approximately 25 to 150) and increase in flexibility for the mutant model I63R in the rest of the residues (about 150 to 260).Increase in the flexibility could make the protein more flexible, and decrease in the flexibility makes protein more rigid.Conformational changes are required for many protein functions [64][65][66], but the conformational flexibility and rigidity must be well balanced [67].The flexibility of all the three mutant models of CDK7 protein is heterogeneous in comparison with the native protein.Thus, from the RMSD and RMSF analysis, it is confirmed that substitution of amino acid adversely affected the stability and flexibility of CDK7 proteins.Beside the different electrostatic interactions, the hydrogen bonds and the salt bridges across the binding interfaces and in the protein interiors serve as main contributors in maintaining the protein structural conformation.Furthermore, incorporation of deleterious nsSNP might change the original electrostatic formations and distances that could affect the protein native structure.Consequently, CDK7 native protein obtained maximum of around 200 hydrogen bonds in the 10 ns simulation period.The mutant models I63R, H135R, and T285M obtained less hydrogen bonds approximately 195 and 190, respectively.The decrease in the number of hydrogen bonds may affect the protein structure.In salt bridge analysis, both the native and mutant models of CDK7 protein maintained the different patterns of salt bridge distances.Changes in the salt bridge distances reflect the displacement of cationic or anionic side chain residues in mutant models.In conclusion, we observed change in bonding distance by hydrogen bonding and salt bridge analysis.Change in residue distance might lead to the loss of thermodynamic stability.The main aim of this study is to extrapolate the relationship between the nsSNPs and their effects in drug-binding capability.In docking analysis, several factors involved between protein-ligand interactions were analyzed, and the analysis revealed the less binding ability of mutant models.In particular, electrostatic potential showed substantial agreement with MD analysis.In conclusion, the given in silico tools can indicate possible deleterious nsSNPs in CDK7 protein.Then, MD studies support the structural and conformational changes for the CDK7 deleterious nsSNP incorporated model.Finally, the binding ability of mutant model with the drug was validated to facilitate the study of new drug-targets and discovery of new drugs for CDK7 protein.In silico approaches reviewed here generated not only a considerable amount of valuable data but also the need for further validation by experimental methods such as in vitro binding/activity assays. ", "section_name": "Discussion", "section_num": null }, { "section_content": "", "section_name": "Materials and methods", "section_num": null }, { "section_content": "The ability to distinguish pathogenic and benign variants from a pool of data is a daunting task.Recently, many computational algorithms have been developed for the feasible prediction of disease-associated variants.Some of the methods classify deleterious variants according to the predicted pathogenicity, and other methods predict the deleterious variants based on protein-stability changes upon mutation.We used both these approaches to identify deleterious variants in the CDK7 gene.Sequence evolutionary information-based methods (SIFT, PANTHER, and PhD-SNP) and the combination of protein structural and functional parameter-based methods (PolyPhen2, I-Mutant3, SNAP, and SNPs&GO) are some of the most reliable tools used for deleterious nsSNP prediction.SIFT, PANTHER, PhD-SNP, SNAP, SNPs&GO, and I-Mutant3 give results in two prediction categories, either tolerated or deleterious, while PolyPhen2 gives results in three categories: benign (probably lacking any phenotypic effect), possibly damaging, and probably damaging (should affect protein function).Sequence-based prediction includes all types of effect at the protein sequence level and can be applied to any human protein with known relatives.Structure-based approach is feasible to implement for proteins with 3D structures.Analyzing deleterious nsSNPs by both sequence and structure level has the added advantage of being able to assess the reliability of the generated prediction results by cross-referencing the results from both approaches.SIFT predicts whether an amino acid substitution affects protein function based on sequence homology and the physical properties of amino acids.A SIFT score ≤0.05 indicates that the amino acid substitution is intolerant or deleterious, whereas a score ≥0.05 is predicted as tolerant [68,69].PANTHER estimates the likelihood of a particular nsSNP causing a functional impact on the protein.PAN-THER uses HMM-based statistical modeling methods and multiple sequence alignments to perform evolutionary analysis of coding nsSNPs.PANTHER subPSEC scores vary from 0 (neutral) to about -10 (more likely to be deleterious).Protein sequences having subPSEC values ≤-3 is said to be deleterious.PolyPhen2.0uses sequence, phylogenetic, and structural information in characterizing the deleterious substitution.A mutation is classified as 'probably damaging' if the probabilistic score is above 0.85 to 1; mutation is classified as 'possibly damaging' if the probabilistic score is above 0.15 to 0.84; the remaining mutations are classified as benign.I-Mutant3 is an SVM-based method for the automatic prediction of protein stability changes upon a single point mutation.The output file shows the predicted free energy change (DDG) which is calculated from the unfolding Gibbs free energy change of the mutated protein minus the unfolding free energy value of the native protein (Kcal/mol).DDG >0 means that the mutated protein has high stability and vice verse.PhD-SNP is a single sequence SVM method (SVM sequence) that discriminates diseaserelated mutations based on the local sequence environment of the mutation at hand and a sequence-profile-based SVM.The tool aims to predict whether an nsSNP causing a single point protein mutation would be a neutral polymorphism or one that is deleterious.SNPs&GO is a method based on SVMs, which predicts disease-associated mutations from protein sequence, evolutionary information, and functions as encoded in the gene ontology terms.The use of functional GO terms is the main aspect of novelty of this tool over other existing bioinformatics tools. From the output of the programs, we only took the binary prediction (pathogenic/neutral) into consideration without taking into account any confidence values provided by some of the programs.SNAP is based on neural network and advanced machine-learning approach to predict the functional effects of nsSNPs in proteins.It uses sequence, functional and structural (secondary structure, solvent accessibility) annotations, and biophysical and evolutionary (residue conservation within sequence families) characteristics to predict a gain or loss in protein function.SNAP predicts whether the mutation is neutral or non-neutral with expected accuracy. ", "section_name": "Computational methods for finding deleterious variants", "section_num": null }, { "section_content": "Protein-ligand interaction study was performed between the native and mutant models of CDK7 protein with the inhibiting compound, flavopiridol.In order to carry out the docking analysis, we used the AutoDock4 suite as a molecular-docking tool.AutoDock4 is a suite of programs making it possible to predict how ligands bind to large macromolecules.In this docking simulation, we used semi-flexible docking protocols.Throughout the docking simulation, the target protein is kept rigid.The ligand being docked is usually flexible and, therefore, explores an arbitrary number of torsional degrees of freedom in addition to the six spatial degrees of freedom spanned by the translational and rotational parameters.AutoDock4 provides different optimization algorithms to search the space of possible protein-ligand combinations, such as simulated annealing, genetic algorithm (GA), and hybrid evolutionary algorithms EA termed Lamarckian GA (LGA) combining the GA with a local search strategy [70]. The Lamarckian Genetic Algorithm (LGA) was chosen to search for the best conformers.The best docking solution (minimum docked free energy) is reported by AutoDock for each GA run.The total number of clusters and the rank of each docking mode (cluster rank) are also reported in the cluster analysis performed by AutoDock.Docking modes were selected on the basis of two criteria: extent of ligands associations with the key residues of the receptor and the thermodynamic stability of the docked complex so obtained.The lowest energy docking mode that would conform to the above said two parameters was selected from over 10 GA runs and hence 10 total docking mode times.The grid boxes were centered on the root of macromolecule with spacing of 0.375 Å.The estimated binding free energies were calculated using the following equation: The unbound structure of ligand is the same as the bound state (crystal structure), so the E internal is equal to E unbound , and they do not contribute to the total energy.On the other hand, the E torsional is calculated based only on the number of torsional bond in ligand, so this term remains the same in each complex.It is clear that there are significant differences between nine charge methods in the estimated binding free energies, so the difference should come from the E intermolecular , including energies of dispersion/ repulsion, hydrogen bonding (hbond), desolvation potential, and electrostatic interactions.The energy functions used in docking simulations attempt to account for the intermolecular energies between the protein and the ligand, as well as the intramolecular energies arising from the ligand conformation itself.AutoDock4 uses a grid-based approach to approximate the energy calculations used by the energy function.During the evaluation of a candidate conformation, the grids were used as lookup tables which store the values used in the calculation, thus making the overall docking simulation exceptionally fast.The Graphical User Interface program ' Auto Dock Tools' was used to prepare, run, and analyze the docking simulations.Kollman united atom charges, solvation parameters, and polar hydrogens were added into the receptor PDB information for the preparation of protein in docking simulation.Gasteiger charges were added in the ligand PDB file. In addition to the Autodock4 study, we used PatchDock for docking native and mutant CDK7 proteins with the drug flavopiridol.PatchDock performs docking based on molecular shape representation and surface patch matching plus filtering and scoring.PatchDock is more reliable because of its fast transformational search, which is driven by local feature matching rather than brute force searching for the six-dimensional transformation space.It further speeds up the computational processing time by utilizing advanced data structures and spatial pattern detection techniques, such as geometric hashing and pose clustering.Protein and the ligand molecule were given as input in performing the docking experiments with default root-mean-square deviation (RMSD) value (4.00 Å).It generated several complex structures based on docking scores.The complex structure file, with the best docking score was selected for further analysis.The geometry of both wild type and mutant type CDK7 structures were optimized through Steepest Descent method with 1000 steps each of GROMACS 4.5.3package.Each minimization was carried out with GROMOS-96 [71] 43a1 force field. ", "section_name": "Protein-ligand docking analysis", "section_num": null }, { "section_content": "Molecular dynamics simulations for the native and mutant models were done with MD simulation package GROMACS 4.5.3 that adopts GROMOS96 43a1 force field parameter for energy minimizations.Energy minimized structures of the native CDK7 and three mutant models were used as a starting point for MD simulations.All the proteins were solvated in a cubic box with wall extending at least 0.9 nm from all atoms and filled with SPC [72] water molecules.A periodic boundary condition was applied that the number of particles, pressure and the temperature were kept constant in the system.In order to obtain electrically neutralized system, we utilized GENION procedure from the GROMACS package to replace random water molecule with Na + or Cl -ions.The temperature was kept constant by using a Berendsen algorithm [73] with a coupling time of 0.2.The minimized system was equilibrated for 10,000ps each at 300 K by position restrained molecular dynamics simulation in order to soak the water molecules into the macromolecules.The equilibrated systems were then subjected to molecular dynamics simulations for 10 ns each at 300 K.In all simulations, the temperature was kept constant at 300 K.The particle mesh Ewald method [74] was used to treat long-range Coulombic interactions and the simulations performed using the SANDER module [75].The SHAKE algorithm was used to constrain bond lengths involving hydrogen's permitting a time step of 2fs.The coordinates were saved at regular time intervals of 1ps.The van der Waals force was maintained at 1.4 nm, and Coulomb interactions were truncated at 0.9 nm. ", "section_name": "Molecular dynamics simulation protocol", "section_num": null }, { "section_content": "Structural properties of the native and mutant models of CDK7 protein were calculated from the trajectory files with the built-in functions of GROMACS 4.5.3The trajectory files were analyzed through the use of g_rmsd and g_rmsf GROMACS utilities in order to obtain the RMSD and RMSF values.The number of distinct hydrogen bonds formed in the protein during the simulation was calculated using g_hbond utility.The number of hydrogen bond was determined on the basis of donor-acceptor distance less than 3.9 nm and of donor-hydrogen-acceptor angle larger than 90° [76].Salt bridge formed in CDK7 protein was analyzed using g_salt GROMACS.If the distance is ≤4.0 nm, the pair is counted as a salt bridge [77].In order to generate the three-dimensional backbone RMSD, RMSF of carbon alpha-carbon, hydrogen bond and salt bridge analysis, and motion projection of the protein in phase space of the system were plotted for all four simulations using Graphing, Advanced Computation and Exploration program. ", "section_name": "Analysis of molecular dynamics trajectories", "section_num": null } ]	[ { "section_content": "This work was supported by the Research Grants Council of Hong Kong (212111) and Faculty Research Grants of Hong Kong Baptist University (3011299), and partially supported by National Natural Science Foundation of China (91029301, 61134013 and 61072149), the Chief Scientist Program of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (2009CSP002), Shanghai Pujiang Program, and the FIRST program from JSPS initiated by CSTP.The authors take this opportunity to thank the management of VIT University for providing the facilities and for encouragement to carry out this work. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "Additional file 1: Figure S1.Secondary structural elements changes in mutant models of CDK7 protein.Additional file 2: Table S1.Involvement of cation-Pi interaction in wild type and mutant structure of CDK7 protein. Additional file 3: Table S2.Flavopiridol interacting residues with CDK7 wild type and mutant type structures. Additional file 4: Figure S2.LIGPLOT analysis of CDK7-flavopiridol complex in both native and mutant states. The authors declare that they have no competing interests. Authors' contributions NN, CGPD, CC, HZ, and LC were involved in the design of the study, drafting the manuscript, and the acquisition, analysis, and interpretation of the data.HZ, CC, and LC supervised the entire study.CGPD, CC, and HZ were involved in the final drafting of the manuscript.All authors read and approved the final manuscript. ", "section_name": "Additional files", "section_num": null }, { "section_content": "Additional file 1: Figure S1.Secondary structural elements changes in mutant models of CDK7 protein.Additional file 2: Table S1.Involvement of cation-Pi interaction in wild type and mutant structure of CDK7 protein. Additional file 3: Table S2.Flavopiridol interacting residues with CDK7 wild type and mutant type structures. Additional file 4: Figure S2.LIGPLOT analysis of CDK7-flavopiridol complex in both native and mutant states. ", "section_name": "Additional files", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. Authors' contributions NN, CGPD, CC, HZ, and LC were involved in the design of the study, drafting the manuscript, and the acquisition, analysis, and interpretation of the data.HZ, CC, and LC supervised the entire study.CGPD, CC, and HZ were involved in the final drafting of the manuscript.All authors read and approved the final manuscript. ", "section_name": "Competing interests", "section_num": null } ]
10.1038/s41598-023-33031-3	Deciphering the spectrum of cutaneous lymphomas expressing TFH markers	<jats:title>Abstract</jats:title><jats:p>T-follicular helper (TFH) markers are expressed in the microenvironnement of marginal zone B-cell lymphoma (MZL), and in lymphomas arising from TFH-cells, sometimes making the differential diagnosis difficult. In the skin, the “TFH-spectrum” is poorly defined, going from primary cutaneous lymphoproliferative disorder with small/medium CD4+ T-cells (SMLPD) to cutaneous localizations of systemic angioimmunoblastic T-cell lymphoma (cAITL), and may pass through intermediate forms (primary cutaneous T-follicular helper derived lymphoma, not otherwise specified (PCTFHL,NOS)). We retrospectively analyzed 20 MZL, 13 SMLPD, 5 PCTFHL, and 11 cAITL clinically, histologically, and molecularly, to define tools to differentiate them. Characteristics that might favor the diagnosis of MZL over SMLPD are: multiple skin nodules (p < 0.001), nodular architecture (p < 0.01), residual germinal centers with follicular dendritic cell network (p < 0.001), monotypic plasma cells (p < 0.001), and few staining with PD1 (p = 0.016) or CXCL13 (p = 0.03). PCTFHL and cAITL presented as multiple (p < 0.01) lesions, in older patients (p < 0.01), with systemic symptoms and/or biological alterations (p < 0.01). Immunophenotypic loss of T-cell markers (p < 0.001), BCL6 (p = 0.023) and/or CD10 staining (p = 0.08), and a higher proliferative index (≥ 30%, p = 0.039) favoured these diagnoses over SMLPD. Pathogenic variants were observed by genomic sequencing in 47% of MZL (<jats:italic>TNFAIP3</jats:italic> (32%), <jats:italic>EP300</jats:italic> (21%), <jats:italic>NOTCH2</jats:italic> (16%), <jats:italic>KMT2D</jats:italic> (16%), <jats:italic>CARD11</jats:italic> (10.5%)), 8% of SMLPD (<jats:italic>TET2</jats:italic>), 40% of PCTFHL (<jats:italic>SOCS1</jats:italic> (20%), <jats:italic>ARID1A</jats:italic> (20%)) and 64% of cAITL (<jats:italic>TET2</jats:italic> (63.6%), <jats:italic>RHOA</jats:italic> (36.4%), <jats:italic>NOTCH1</jats:italic> (9%)). This study characterizes the various clinical and histological features between cutaneous lymphomas expressing TFH markers and highlights the value of the interest of screening for genomic mutations in difficult cases.</jats:p>	[ { "section_content": "entities which present a distinct aggressiveness and evolution.Furthermore, molecular alterations of PCTFHL are unknown. TFH-cells are also find in the microenvironnement of B-cell lymphomas, especially marginal zone lymphomas (MZL) 6,[8][9][10] .The clinicopathological characteristics of cutaneous MZL are close to some cutaneous T-cell lymphomas with a TFH phenotype, in particular primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder (SMLPD) 10 .The distinction between MZL and SMLPD is sometimes difficult and clonality study is not always discriminatory due to cases without monoclonal rearrangement or with both B (BCR) and T-cell receptor (TCR) rearrangements 7 . Targeted Next Generation Sequencing (TNGS) has proven its interest in daily practice 11 , but few studies have analyzed the molecular profile of cutaneous lymphomas.Concerning AITL, most studies focus on nodal locations, in which mutations of TET2 (52-76%), IDH2 (20-45%), DNMT3A (30-40%) and RHOA G17V (28-70%) are typical [11][12][13][14][15][16] .The only study currently published in cutaneous localizations of AITL is the one of Leclaire Alirkilicarslan et al., which included 41 patients and found IDH2 R172K/S and RHOA G17V mutations in 19% and 78% of cases respectively 17 .A recent study also showed a mutation of DNMT3A 18 in one case of SMLPD. We retrospectively described a series of 49 cutaneous lymphomas with TFH-markers expression, including cases with TFH expression in tumoral cells and cases with TFH hyperplasia in the microenvironnement, to characterize them at a clinicopathological and molecular level and highlight tools to differentiate them. ", "section_name": "", "section_num": "" }, { "section_content": "Patient selection.TFH-cells arising lymphomas (SMLPD and cutaneous localizations of systemic AITL) and cutaneous MZL diagnosed from March 2017 to March 2019 in agreement to the 2016 WHO classification of hematologic malignancies were retrieved from the pathological department of the Lyon Sud University Hospital, France.Intermediate forms between SMLPD and AITL, so-called PCTFHL, were also retrieved. Inclusion criteria for MZL, PCTFHL and SMLPD were the presence of a monoclonal B-or T-cell population, to limit the inclusion of reactive lymphoid hyperplasia, and enough FFPE material available.All cases had to have benefited from an extensive assessment including either a CT-scan or chest X-ray and abdominal and pelvic ultrasound, to exclude systemic lymphoma with secondary involvement of the skin.MF and/or Sezary syndrome were ruled out by clinicopathological correlation and expert opinion from the French cutaneous lymphomas study group (GFELC).Concerning cutaneous localizations of AITL, cases were included only if they had a known history of systemic AITL, proved by a lymph node biopsy. Clinical, biological, and radiological data.Clinical data were retrospectively collected.It included: age at diagnosis, sex, medical history, clinical presentation (number of lesions, localization, systemic symptoms, and lymphadenopathy), biological data (blood count, borreliosis serology), imaging data, received treatments and evolution.Patients were divided into two groups: (i) \"indolent\" (spontaneous disappearance and/or no relapse after local treatment); or (ii) \"refractory/relapsing\" (relapse/persistence after initial local treatment requiring chemotherapy or radiotherapy). ", "section_name": "Methods", "section_num": null }, { "section_content": "Experts pathologists of the French cutaneous lymphomas study group (GFELC) [18][19][20] (BB, OH) or of the Lymphopath Network 21 (ATG, MD) reviewed cases.They recorded: localization (superficial, mid or deep-dermis, hypodermis), architecture (lichenoid (sub-epidermal band), nodular, diffuse, or interstitial (scattered cells in the dermis) pattern), size of cells, epidermotropism (in these cases, results of the blood immunophenotyping were retrieved to exclude cases with a doubtful diagnosis with Sezary syndrome), germinal centers, and associated cells. Immunochemistry study.Immunohistochemistry for CD2, CD3, CD4, CD5, CD7, CD8, TFH markers, CD20, CD21, CD23, CD30, CD138, kappa, lambda, and Ki67/Mib1 was performed using standard procedures (Leica BOND-MAX, Leica Microsystems SA, Nanterre, France).In situ hybridization for Epstein-Barr virus (EBV) encoded RNAs (EBER) was performed using a Ventana BenchMark XT automated immunostainer.Protocol and antibodies are detailed in Suppl.Methods and Suppl.Table S1.The proportion of B and T-cells was estimated as the ratio of cells to the total lymphocytic infiltrate.The expression of CD138 was scored semi quantitatively on the basis of the proportion of positive cells in the whole tissue: 0: no staining, 1+: < 10%, 2+: 10 to 25%, 3+: ≥ 25%.Immunochemistry of TFH markers was performed using the following antibodies: PD1 (NAT105; Roche Diagnostics; Meylan, France), CXCL13 (BCA-1; R&D System, Minneapolis, USA), BCL6 (GI191E/A8; Roche Diagnostics; Meylan; France), CD10 (SP67; Roche Diagnostics; Meylan; France), and ICOS (AB105227; Abcam, Cambridge, UK).Their expression was scored quantitatively on the basis of the proportion of positive cells in the whole T-cells infiltrate.They are presented with their standard deviation.The proliferative index was evaluated as the percentage of Ki67-positive cells relative to the total lymphocyte population. ", "section_name": "Histological review.", "section_num": null }, { "section_content": "Clonality study was performed according to the EuroClonality/BIOMED-2 protocol 22 .IGH and IGK assays were used for BCR study, with the following primers: FR1, FR2, FR3, DH-JH, Ig kappa, and Ig lambda.TCR Clonality was assessed using the BIOMED-2 primer sets for TCR gamma (TCRG).TNGS have been performed as previously reported 11,23 .The applied panel of 47 genes is detailed in Suppl.Table S2.Only pathogenic variants with a minimal depth of 700× were retained.Supplemental quality data are available in Suppl.Table S4. ", "section_name": "Clonality study and targeted next generation sequencing (TNGS).", "section_num": null }, { "section_content": "Statistical analysis has been performed using the Medistica pvalue.iosoftware 24 , performing univariate analysis for descriptive variables.Kruskal-Wallis test was used to study ordinal qualitative ordinal variables (immunohistochemistry results) and quantitative variables (age) in the 4 independent groups (CMZL, SMLPD, PCTFHL, and cAITL).Fisher's exact test or χ 2 test were used to study nominal qualitative variables (clinical and histological characteristics) in the different groups. Compliance with ethical standards in research.This study was performed in accordance with the Declaration of Helsinki.Informed consent was obtained from all subjects and/or their legal guardian(s) according to the guidelines of the French Bioethics Law.The protocol has received the validation of the local ethics committee (Ethics committee of the Hospices Civils de Lyon, number 21_5588). ", "section_name": "Statistical analysis.", "section_num": null }, { "section_content": "Sixty-seven cases corresponding to 27 MZL, 21 SMLPD, 8 PCTFHL and 11 cAITL were retrieved.Seven MZL were excluded due to the presence of a systemic MZL.Eight SMLPD were excluded: 3 because the material was exhausted, 4 due to the absence of any monoclonal T-cell population, and one because further evaluation led to reclassification as MF.Three PCTFHL were excluded, two due to the absence of TCR clonal rearrangement, and one due to the presence of Sezary cells in the blood, leading to reclassification as Sezary syndrome.Twenty MZL, 13 SMLPD, 5 PCTFHL, and 11 cAITL were finally included.SMLPD presented as unique (100%) erythematous nodule (9/13, 69%) or erythematous-squamous plaque (4/13, 31%), mainly located on the trunk (55%) or head and neck (30%), with a median age of 56-years-old [25-78].Borrelia serology was negative in all cases.Blood counts were normal. ", "section_name": "Results", "section_num": null }, { "section_content": "PCTFHL presented as multiples (5/5, 100%) erythematous-squamous plaques (2/5, 40%) or erythematous nodules (2/5, 40%), or papules (1/5, 20%).One case (PCTFHL no 5) presented a monoclonal circulating CD4+ T-cell population without immunophenotyping feature of Sezary syndrome.The median age was 74-yearsold [47-91].Two PCTFHL presented biological alterations (anemia: Haemoglobin 11.2 g/dl and increased LDH level: 352 U/L). Most cAITL presented as a maculopapular rash (6/11, 55%), with a median age of 74-years-old [56-92].Other cases presented as multiple (100%) erythematous papules (2/11, 18%) or erythematous-squamous plaques (3/11, 27%).In AITL no 11 the disease appeared as a unique, infiltrated, necrotic plaque of the abdomen.In six cases, biopsies were performed at the onset of the disease (at diagnosis).For the five other cases, cutaneous lesions occurred during a relapse of the disease (after a first line of chemotherapy).All patients described systemic symptoms (deterioration in the general condition: n = 11/11, pruritus: n = 3/11, fever: n = 4/11) or biological alterations (bi/pancytopenia: n = 7/11, increase in LDH [338-600]: n = 4/11). Histological review and immunohistochemistry study (Table 2).MZL (Figs. 1,2) presented a nodular architecture (19/20, 95%), with residual germinal centers (GC) (12/20, 60%) and a CD23+ follicular dendritic cell (FDC) network (15/20, 75%).One case presented a lichenoid architecture.Hypodermis involvement was obseved in 7/20 cases (35%).MZL were composed of more B (55% (± 17.2)) than T-cells (43.5% (± 16.1)), but 4 had more T-cells than B-cells, and 2 had equal proportions of both.CD138+ plasma cells were present in almost all cases (n = 19/20, 95%), representing more than 10% of cells in 65% of cases, and being monotypic in 75% of cases.Eosinophils were present in 4/20 cases (20%).TFH markers were not constantly expressed in MZL cases; PD1: n = 18/20; CXCL13: n = 15/20; BCL6: n = 5/20; no CD10 expression, and average of stained were low; PD1: 19% (± 17.6); CXCL13: 8% (± 8.01); BCL6: 2.5% (± 4.44).ICOS was expressed in 6 cases (< 1% of stained cells).When positive, PD1 was expressed with a high intensity on the T-cells of the germinal centers, and, with a weak or moderate staining in small T-cells of the background.Ki67 was evaluated at 18% (± 6.1).SMLPD (Fig. 3) presented a nodular (7/13, 54%), diffuse (2/13, 15%), or lichenoid (4/13, 31%) pattern, without residual GC nor FDC network, and with hypodermis involvement in 6/13 cases (46%).Eosinophils were present in 6/13 cases (46%).Infiltrate was composed of a majority of T (61.5% (± 11.4)) rather than B-cells (35% (± 12)), except in 4 cases in which T and B-cells were equally distributed.There was no loss of T-cell markers.Plasma cells were present (n = 12/13, 92%), but represented less than 10% of cells in most cases (n = 11/12, 85%) and were always polytypic.PD1 and CXCL13 were seen in all cases, BCL6 in 6 cases and ICOS in 4 cases.Average of stained T-cells were; PD1: 49% (± 33.7), CXCL13: 20% (± 20.0), BCL6: 11% (± 20.7), ICOS: < 1%.There was no CD10 expression.PD1 showed a higher intensity in medium/large cells, and some PD1+ cells tended to form clusters or \"rosettes\" around large lymphocytes (Fig. 3H).Ki67 was evaluated at 15% (± 6.6). In PCTFHL (Fig. 4), architecture was lichenoid (3/5, 60%), interstitial (1/5, 20%) or nodular (1/5, 20%).There were no epidermotropism, excepted in PCTFHL no 3. Hypodermis involvement was rare (1/5, 20%).Eosinophils were seen in 3 PCTFHL (60%).There were no residual GC nor FDC network.Plasma cells were rare (n = 2/5, 40), representing less than 10% of the cells without monotypia.T-cell markers were lost in 80% of PCTFHL (CD7: 3/5, CD5: 1/5).PD1, CXCL13 and BCL6 were expressed in all cases, and CD10 expression was observed in 2/5 PCTFHL.Average of stained T-cells in PCTFHL were; PD1: 71% (± 21.3); CXCL13: 30% (± 20.0); BCL6: 20% (± 12.2), and CD10: 4% (± 5.48).ICOS had not been performed.PD1 was expressed with a moderate to high intensity on the T-cells, with a diffuse pattern.Ki67 was evaluated at 32% (± 27.7). In cAITL (Fig. 5), infiltrates were sparse; architecture was mostly interstitial (9/11, 82%) than nodular (2/11, 18%).Plasma cells were rare (n = 4/11, 36%), representing less than 10% of the cells without monotypia.Eosinophils were seen in 7 cAITL (64%).There were no residual GC nor FDC network.Hypodermis involvement was never seen.CD30+ B-cells were seen in 8/11 cAITL, with overexpression of ARN EBERs using in situ hybridization in 1/11 case.T-cell markers were lost in 45.5% of cAITL (CD7: 4/11, CD5: 1/11).PD1, CXCL13 and BCL6 were expressed in all cases, and CD10 expression was observed in 3/11 cAITL (28%).Average of stained T-cells in cAITL were; PD1: 40.5% (± 19.7); CXCL13: 16.5% (± 16.7); BCL6: 21% (± 17.7), and CD10: 4% (± 6.74).ICOS had not been performed.PD1 was expressed with a moderate to high intensity on the T-cells, with a diffuse pattern.Ki67 was evaluated at 35% (± 22). ", "section_name": "Clinical presentation (Table 1", "section_num": null }, { "section_content": "Both CMZL and SMLPD presented as erythematous nodules (80% vs. 69%, p = 0.68), but lesions were more often multiples in MZL (55% vs. 0%, p < 0.001).Histologically, nodular architecture (95% vs. 54%, p < 0.01), presence of residual germinal centers with FDC network (75% vs. 8%, p < 0.001), and monotypic plasma cells (75% vs. 0%, p < 0.001) were in favor of MZL versus SMLPD.Lichenoid architecture was not significantly different in these two subgroups (p = 0.06), nor was the presence of eosinophils (p = 0.14).B-cells were more abundant in MZL (55% vs. 35%, p < 0.01), whereas T-cells were more abundant in SMLPD (61.5% vs. 43.5%,p < 0.001).PD1 expression was lower in MZL compared to SMLPD (19% vs. 49%, p = 0.016).CXCL13 (8% vs. 20%, p = 0.03) and BCL6 (2.5% vs. 11%, p = 0.27) were less expressed in CMZL than SMLPD.Table 1.Clinical data of patients with MZL, SMLPD, PCTFHL, and cutaneous location of AITL including clinical presentation, treatment, and evolution.Locations of lesions are the total number of lesions in the location section is higher than the number of patients, as some patients had multiple lesions in different locations.NA: not analyzed, MZL: marginal zone lymphoma, SMLPD: primary cutaneous (CD4+) small/ medium T-cell lymphoproliferative disorder; PCTFHL: primary cutaneous T-follicular helper derived lymphoma; cAITL: cutaneous localization of angio-immunoblastic T lymphoma.Significant values are in bold.ent within the spectrum of TFH lymphomas (p = 0.71).Losses of T-cell markers were seen only in PCTFHL (80%) and cAITL (45%) (< 0.001).PD1 was more often expressed in PCTFHL compared to other subtypes, without significant statistical differences (SMLPD: 49%, PCTFHL: 71%, cAITL: 40.5%, p = 0.12).There were no statistical differences between the expressions of CXCL13 in lymphomas of the TFH spectrum (SMLPD: 20%, PCTFHL: 30%, cAITL: 16.5%, p = 0.79).BCL6 was significantly more frequent in PCTFHL and cAITL than SMLPD (SMLPD: 11%, PCTFHL: 20%, cAITL: 21%, p = 0.024).CD10 was expressed only in PCTFHL and cAITL (p = 0.083).Proliferative index was slightly more elevated in intermediate forms and systemic lymphomas (SMLPD: 15% (± 6.60), PCTFHL: 32% (± 27.7) and cAITL: 35% (± 22.0), p = 0.039). Treatments and evolution (Tables 1,4).MZL treatments consisted of topical corticosteroids (11/20, 55%), doxycycline (10/20, 50%), often associated with surgical excision (15/20, 75%).Nine cases suffered local relapses, treated by topical corticosteroids (6/9), radiotherapy (3/9).SMLPD treatments consisted in topical corticosteroids (betamethasone dipropionate or clobetasol propionate, 3/13, 23%), antifungal cream (1/13, 8%), antibiotic therapy (21 days of doxycycline, 3/13, 23%), alone or associated with surgical excision (10/13, 77%).One case presented a spontaneous disappearance after biopsy.All cases presented an indolent evolution.Among the three cases which did not undergo surgery, two suffered a relapse after dermocorticoids disruption. Among PCTFHL, the median follow-up was of 20 months.The case no 5 with a monoclonal circulating T-cell population responded completely under topical corticosteroids and was considered indolent.Three were refractory/relapsing patients; one received topical corticosteroids with a remitting relapsing course (median G-I) (CD138 × 10, kappa/lambda, ×20); Association to numerous monotypic kappa plasma cells located outside of the B-cell nodules; (J) (PD1, ×10), (K) (CXCL13, ×20), and (L) (BCL6, ×10): small T-cells of the background harbor a TFH phenotype (PD1 30%, CXCL13 15%, BCL6 10%, CD10 negative).Proliferative index using Ki67/Mib1 was evaluated a 20% (picture not shown).The diagnosis of MZL was confirmed thanks to the presence of a monoclonal B-cell population.This case displayed no pathogenic variant in TNGS. follow-up of 50 months), one received radio-chemotherapy (gemcitabine), and the last received methotrexate.The fifth was lost to follow-up. All cAITL were treated by chemotherapy (9 CHOP: cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) and 2 GEMOx: gemcitabine, oxaliplatin). ", "section_name": "Clinical and histological comparison between CMZL and SMLPD.", "section_num": null }, { "section_content": "BCR rearrangements were present in all MZL and TCR rearrangements in all SMLPD and PCTFHL.The same clone was found in the skin biopsy for the PCTFHL case with a circulating T monoclonal population. Concerning cAITL, 5 presented a monoclonal T-cell population, 2 presented both BCR and TCR rearrangement, results were undetermined in 3 cases, and one did not display any monoclonal population (AITL no 9). Two SMLPD and one MZL presented both BCR and TCR rearrangements.The 2 SMLPD presented characteristics typical of SMLPD: unique nodules, 66-80% of T-cells, absence of FDC network, 5-20% polytypic plasma cells, medium PD1/CXCL13 staining (10-30%).The MZL case presented characteristics typical of MZL: multiple lesions, majority of B-cells (60%), nodular pattern, residual germinal centers, 25% of plasma cells with lambda monotypia, low PD1/CXCL13 staining (10%).3,4).The TNGS analysis (Table 3) was performed in all cases except for 1 MZL and 1 SMLPD due to a poor DNA quality.Pathogenic variants were found in 9 MZL (47%), 1 SMLPD (8%), 2 PCTFHL (40%) and 7 cAITL (64%).Pathogenic variants found in MZL were: TNFAIP3 ( quality NGS data, few mutations were identified in SMLPD and PCTFHL.Pathogenic variant found in SMLPD was TET2 (1/12, 8.5%).Pathogenic variants found in PCTFHL were SOCS1 (1/5, 20%) and ARID1A (1/5, 20%).Patient with the SOCS1 variant presented the following characteristics: presentation as multiple erythematous and infiltrated plaques without Sezary cells or circulating population, lichenoid pattern with epidermotropism, 60% of T-cells (CD7 lost) and 40% of B-cells, high expression of TFH markers (PD1 80%, CXCL13 50%, BCL6 20% CD10 10%).Patient with the ARID1A variant presented the following characteristics: multiple papules without Sezary cells or circulating population, lichenoid pattern separated from the epidermis by a grenz-zone and without epidermotropism, 80% of T-cells (CD7 lost) and 20% of B-cells, variable expression of TFH markers (PD1 95%, CXCL13 20%, BCL6 10%, no CD10 expression).Pathogenic variants found in cAITL were: TET2 (n = 7, 64%), RHOA (n = 4, 36%; 3 RHOA G17V and 1 RHOA G17L ), and NOTCH1 (1/11, 9%).Five cAITL displayed at least two pathogenic variant (TET2 ± RHOA or NOTCH1), and two displayed only variants of TET2.Patients with TET2 mutations had two TET2 Single Nucleotide Variant (SNV) except one, with a single TET2 SNV. ", "section_name": "Clonality study (Tables 2, 3, 4). With the presence of a monoclonal population being inclusion criteria,", "section_num": null }, { "section_content": "Molecular analysis had also been performed in addition to the skin tissue in lymph node biopsies of 4 AITL (Table 4).Cases no 3, 7 and 10 presented a similar monoclonal T population both samples, case no 9 had had a TCR gamma chain rearrangement with an undetermined ratio in the skin biopsy, and a true T monoclonal (G) (CD138, ×10): very few associated plasma cells (black arrow), which were polytypic for kappa and lambda, and located outside of the proliferation in this particular case, but scattered among T-cells in most SMLPD cases; (H) (PD1, ×20), (I) (CXCL13, ×20), (J) (BCL6, ×20) and (K) (CD10, ×20): huge expression of TFH markers except for CD10 which was negative (PD1 90%, CXCL13 40%, BCL6 25%).As illustrated, PD1 expression shows a higher intensity in medium/large cells, and some PD1+ cells also tended to form \"rosettes\" around large lymphocytes (insert).Proliferative index using Ki67/Mib1 was evaluated a 15%, and no follicular dendritic cells network (pictures not shown).The diagnosis of SMLPD was confirmed thanks to molecular data; the presence of a monoclonal T-cell population.This case displayed an isolated TET2 pathogenic variant using TNGS.population in the lymph node.Cases no 7, 9 and 10, presented the same pathogenic variants in both samples, including TET2 and/or RHOA G17V hotspot. ", "section_name": "TNGS analysis (Tables", "section_num": null }, { "section_content": "The differential diagnosis of cutaneous lymphomas with TFH expression and/or hyperplasia is frequently a diagnostic challenge in daily diagnosis work.The spectrum of cutaneous T-cell lymphomas arising from TFHcells extends from SMLPD to AITL 6 .Hyperplasia of TFH reactive T-cells is also frequently observe in cutaneous MZL 25 .The objective of the present study was to better characterize these different lesions (20 MZL, 13 SMLPD, 5 PCTFHL, and 11 cAITL) at the clinicopathological and molecular levels and to highlight tools to differentiate them.(D,E) (CD3, ×10, ×20) and (F,G (CD20, ×10, ×20): slight predominance of T-cells (60%), with a partial loss of CD7, associated to numerous B-cells (without CD30 expression or RNA EBER positivity); (H) (PD1, ×20), (I) (CXCL13, ×20), (J) (BCL6, ×20) and (K) (CD10, ×20): huge expression of TFH markers (PD1 80%, CXCL13 50%, BCL6 20% CD10 10%).Proliferative index using Ki67/Mib1 was evaluated a 35% (picture not shown).The diagnosis of PCTFHL was confirmed thanks to molecular data; the presence of a monoclonal T-cell population.This case displayed a pathogenic variant of SOCS1 using TNGS. The clinical presentation and histological appearance of the 49 cases described in this study were consistent with the literature 3,6,18,26 .In particular, MZL corresponded to erythematous papules/nodules (80%), or plaques (20%), which were most frequently located on the limbs (50%) and trunk (40%) 26 .Infiltrates in SMLPD could be separated into two patterns, as described by Beltzung et al.; (i) \"Pattern 1\" (n = 9/13, 69%): erythematous nodules, nodular/diffuse architecture, located on the head and neck (n = 4/9, 44%), followed by the trunk (n = 3/9, 33%) and upper extremities (n = 2/9, 23%); and (ii) \"Pattern 2\" (n = 4/13, 31%): erythematous-squamous plaques, lichenoid architecture, all located on the trunk 18 .Interestingly, loss of CD7 is described in the literature in 24% of SMLPD 18 .However, in this serie, none of the SMLPD cases showed CD7 loss, even partially.Although these data need to be confirmed in a larger cohort, it could be a tool to differentiate more aggressive forms of SMLPD, or even PCTFHL.Clinical presentation of PCTFHL was similar as described (multiple papules, plaques, and nodules of trunk/head) 5,6 , excepted that only one case presented a circulating monoclonal population (vs.4/5 for Battistella et al.) 6 .Maculo-papular rash seems to be the classical presentation of cutaneous locations of AITL 3 . MZL appears to differ clinically from SMLPD in their presentation as multiple skin nodules (p < 0.001).Histologically, nodular architecture (p < 0.01), presence of an FDC network (p < 0.001), and presence of monotypic plasma cells (p < 0.001) are characteristics that should favor the diagnosis of MZL rather than SMLPD.PD1 and CXCL13 were expressed in all SMLPD, but inconstantly in MZL (90% and 75% respectively), and with fewer positive cells in MZL than SMLPD: PD1 (19% in MZL vs. 49% in SMLPD, p = 0.016), CXCL13 (8% in MZL vs. 20% in SMLPD, p = 0.03), or BCL6 (2.5% in MZL vs. 11% in SMLPD, p = 0.27).PD1 expression profile was also different in MZL and SMLPD ; PD1 was expressed on medium to large and frequently clustered cells in SMLPD 18 , whereas, in MZL, PD1 was expressed by the small T-cells of the germinal centers.), (H) (CD10, ×20), (I) (BCL6, ×20) and (J) (CXCL13, ×20): Expression of TFH markers (PD1 40%, CD10 25%, BCL6 10%, and CXCL13 10%).Proliferative index using Ki67/Mib1 was evaluated a 70% (picture not shown).The diagnosis of cAITL was confirmed thanks to molecular data; the presence of a monoclonal T-cell population associated with a minor monoclonal B-cell population.This case did not display any pathogenic variant using TNGS. Table 3. Results of molecular biology analysis: clonality study and Targeted Next-Generation Sequencing (TNGS) put in parallel with clinical evolution of patients and their received treatments.AF: antifungal cream, DLBCL: diffuse large B cell lymphoma, cAITL: cutaneous localization of angio-immunoblastic T lymphoma, CT: chemotherapy, DXC: doxycycline, RT: radiotherapy, MTX, methotrexate, MZL: (cutaneous) marginal zone lymphoma, NA: not available/analyzed, R: rituximab, SE: surgical excision, SMLPD: (Primary cutaneous CD4 +) small/medium T-cell lymphoproliferative disorder, TC: topical corticosteroids, PCTFHL: primary cutaneous T-follicular helper derived lymphoma, UNDET: undetermined.PCR in a cohort of cAITL.In nodal AITL, mutations are much more frequent 11,12,15 ; TET2 SNV (76%), RHOA G17V (60-78%), and IDH2 R172 substitutions (19.5%).In the present study, the percentage of mutated cases was lower than described in the literature, and no IDH2 pathogenic variants were found.This may be explained by the small number of cases in the study or by the low density of tumoral cells in the samples.However, these molecular findings were confirmative of the diagnosis in 64% of cases, as in 4/5 cases with an atypical presentation (slight perivascular infiltrates, no identifiable atypical lymphocytes) in the study of Leclaire Alirkilicarslan et al.These data suggest that TNGS may represent a useful tool for the diagnosis of cAITL.The VAF of the SNV were lower in cAITL (average = 6.7%, median = 3.1%, ranging from 1.2 to 22%), compared to 14.2% in MZL and 45.2% in PCTFHL.It can be explained by the low cell density in these biopsies (less than 25% of the surface) or by clonal tumor heterogeneity, as already described in these entities 13,33 .Nevertheless, the sequencing quality remains optimal; the described variants are robust due to a minimal depth of 700× (Suppl.Table 3). The second objective of this study was to decipher the spectrum of TFH lymphomas, including also lymphoproliferations and controversial intermediate forms.Concerning SMLPD, only one case (8%) presented a pathogenic variant of TET2, which may correspond to clonal hematopoiesis.Beltzung et al. reported a unique pathogenic variant of DNMT3A among 13 SMLPD, but this gene was not part of our panel 18 .Concerning PCT-FHL, two cases (40%) presented isolated pathogenic variants of ARID1A and SOCS1.These two mutated cases required aggressive treatment, whereas the two cases with an indolent disease did not present any detectable variant.ARID1A mutations are reported in numerous cancers and lymphomas and therefore does not seem really helpful in isolation 15,34 .SOCS1 was a class 3 of uncertain clinical significance 35,36 .This variant has already been described in TFH lymphomas 34 , but also in MF 37 .This finding was arguable, PCTFHL currently not being a recognized entity, and of difficult differential diagnosis with MF expressing TFH markers or Sezary syndrome 38,39 .In PCTFHL no 3 in particular, the clinical presentation as erythematosquamous plaques, the presence of epidermotropism, and the SOCS1 mutation may have suggested the diagnosis of MF or Sezary syndrome.Nevertheless, the absence of Sezary cells in the blood, the presence of interface dermatitis lesions, the polymorphism of the infiltrate (presence of 50% of B-cells and numerous plasma cells), and the intense expression of all TFH markers (PD1: 80%, CXCL13: 50%, BCL6: 20%, CD10: 10%) pleaded against this diagnosis.Within the panel studied, there does not appear to be a common or recurrent molecular profile between SMLPD, PCTFHL, or cAITL.The molecular abnormalities usually present in AITL were not found in SMLPD or PCTFHL.These data remain debatable and future studies will undoubtedly allow a better classification of these case.Indeed, one of the raised hypotheses was that the spectrum of TFH-lesions was potentially underpinned by a mutational spectrum.The frequency of mutations found in TNGS (8% in SMLPD, 40% in PCTFHL and 64% in cAITL) seems to go in the same direction as the prognosis of these lesions.As already described in nodal AITL, these data may suggest that RHOA mutation may be a secondary event in lymphomatous cells after other critical molecular events such as TET2 mutations that first occur in \"premalignant\" lymphocyte precursors 13,17,[40][41][42] .This hypothesis may agrees with the distribution of pathogenic variants in the present study and reinforces the theory of a spectrum of lymphoma derived from a TFH lymphocyte.These data will need to be confirmed in a larger cohort, gathering a larger number of PCTFHL cases.If accepted, the provisional PCTFHL entity will also need to be defined, in particular its exact terminology (lymphoproliferation or lymphoma), and diagnosis criteria.The controversial nature of these intermediate forms makes it difficult to include these patients in studies. This study highlights some histological features to distinguish the main subtypes of cutaneous lymphomas expressing TFH markers, that might reasonably be considered in a differential diagnosis.It also underlines the interest of integrated histomolecular diagnosis, using clonality and NGS, to classify these pathologies and strength the hypothesis of a spectrum of cutaneous lymphomas arising from a T-follicular helper lymphocyte is, even if further studies on a more significant number of patients are required to draw firm conclusions.In particular, the provisional entity so-called PCTFHL will need to be defined thanks to larger cohorts, from clinical, histological, and molecular points of view, in order to determine if it related to lymphoproliferations (such as SMLPD), or to real lymphomas with TFH phenotype, by analogy to nodal lymphomas. ", "section_name": "Discussion", "section_num": null } ]	[ { "section_content": "Authors would thank Laurianne Brand, Blandine Grangier, Aurélie Gaultier, and Mathilde Bardou (Centre de Pathologie et de Biologie Moléculaire de Lyon Sud, Hospices Civils de Lyon) for their excellent technical support. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "All data generated or analyzed during this study are included in this published article (and its supplementary information files). ", "section_name": "Data availability", "section_num": null }, { "section_content": "www.nature.com/scientificreports/ Concerning T-cell lymphoproliferations/lymphomas, features in favor of a more aggressive disease such as PCTFHL or AITL are: elderly patients (p < 0.01), presentation as multiple (p < 0.001) erythematous and scaly patches or as a maculopapular rash for cAITL (p < 0.001), association with systemic symptoms and/or biological alterations (p < 0.001), interstitial architecture (p < 0.001), loss of T-cell markers (p < 0.001), and an elevated proliferative index (p < 0.01).Concerning TFH markers, there was no difference in the amount of stained cells with PD1 (p = 0.12) or CXCL13 (p = 0.79) across these subtypes, but BCL6 (p = 0.024) and CD10 (p = 0.08) seemed more often positive in cAITL and PCTFHL.Extension assessment, including a CT scan and immunophenotyping of the circulating blood, is particularly important to differentiate PCTFHL from cAITL. In this study, the main objective was to compare these entities.That's why it was decided to include only cases with a monoclonal population, to try to include only \"typical\" cases and to limit the possibility of including inflammatory diseases, as, in the skin, dominant clones may also be found in a variety of benign dermatoses.Literature has already proved the interest of clonality assessment in the diagnosis of cutaneous lymphomas, the finding of a monoclonal population orienting the diagnosis in more than 80% of MZL 26 , more than 65% of SMLPD 18,27 , and all PCTFHL 6 .This study further emphasizes the importance of an integrated histomolecular diagnosis.Indeed, 4 MZL presented a PD1 expression with more than 30% of stained cells, making the differential diagnosis with SMLPD particularly difficult without molecular data.In these patients, a monoclonal B-cell population was found, without monoclonal T-cell population, and the diagnosis of MZL could be made.The study of clonality may therefore be helpful in these cases of MZL with TFH hyperplasia of the microenvironnement 10 .Moreover, this study also highlights the value of comparing molecular techniques in cases of suspected cutaneous localization of systemic lymphoma.discovery of the same monoclonal population (AITL no 3) or same NGS pathogenic variants (AITL no 7, 9 and 10), in skin biopsy and lymph node can, indeed, allow to link two locations of the same disease. Taken individually, TNGS also identified pathogenic variants helpful for the diagnosis in 42% of MZL and 64% of cAITL.Even if these cases do not represent a large majority, they are not negligible considering the importance of an optimal classification of these lesions for the management and prognosis. In the present study, 47% of MZL presented pathogenic variants, involving TNFAIP3 (n = 6, 32%), KMT2D (n = 3, 16%), EP300 (n = 4, 21%), NOTCH2 (n = 3, 16%), and CARD11 (n = 2, 10.5%).According to the Cosmic database, NOTCH2 (19%), KMT2D (15%), and TNFAIP3 (11%) are the top 3 most frequent mutated genes observed in mucosa-associated lymphoid tissue (MALT) lymphomas 28 and nodal MZL 29 .EP300 and CARD11 are also frequently described in MZL 30 .FBW7 has not been considered as helpful 31 .Mutations do not seem correlated to prognosis in MZL: 44% of indolent cases among the mutated cases and 60% of relapses among the non-mutated cases.However, the panel targeted the most relevant genes for lymphoma diagnosis but did not cover all exons for some genes (e.g., KMT2D) and lacked a few select genes (e.g., FAS, SLAMF1, SPEN, and NCOR2), which are described in cutaneous MZL 10,32 .This could explain the negativity of some cases and the lack of correlation with the clinical course. Concerning AITL, most studies concern nodal locations, in which mutations of TET2 (52-76%), IDH2 (20-45%), DNMT3A (30-40%) and RHOA G17V (28-70%) seem frequent [11][12][13][14][15][16] .The only study currently published in cutaneous localizations is the one of Leclaire Alirkilicarslan et al., which included 41 patients and found IDH2 R172K/S and RHOA G17V mutations in 19% and 78% of cases respectively using PCR 17 .This study is the only one to study mutations using TNGS on a cohort of cutaneous localizations of AITL.Among them, 64% presented pathogenic variants involving TET2 (n = 7, 64%), RHOA (n = 4, 36%), and NOTCH1 (n = 1, 9%).Leclaire Alirkilicarslan et al. found 78% of RHOA G17V and 19% of cases with IDH2 R172 substitutions using The authors declare no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "www.nature.com/scientificreports/ Concerning T-cell lymphoproliferations/lymphomas, features in favor of a more aggressive disease such as PCTFHL or AITL are: elderly patients (p < 0.01), presentation as multiple (p < 0.001) erythematous and scaly patches or as a maculopapular rash for cAITL (p < 0.001), association with systemic symptoms and/or biological alterations (p < 0.001), interstitial architecture (p < 0.001), loss of T-cell markers (p < 0.001), and an elevated proliferative index (p < 0.01).Concerning TFH markers, there was no difference in the amount of stained cells with PD1 (p = 0.12) or CXCL13 (p = 0.79) across these subtypes, but BCL6 (p = 0.024) and CD10 (p = 0.08) seemed more often positive in cAITL and PCTFHL.Extension assessment, including a CT scan and immunophenotyping of the circulating blood, is particularly important to differentiate PCTFHL from cAITL. In this study, the main objective was to compare these entities.That's why it was decided to include only cases with a monoclonal population, to try to include only \"typical\" cases and to limit the possibility of including inflammatory diseases, as, in the skin, dominant clones may also be found in a variety of benign dermatoses.Literature has already proved the interest of clonality assessment in the diagnosis of cutaneous lymphomas, the finding of a monoclonal population orienting the diagnosis in more than 80% of MZL 26 , more than 65% of SMLPD 18,27 , and all PCTFHL 6 .This study further emphasizes the importance of an integrated histomolecular diagnosis.Indeed, 4 MZL presented a PD1 expression with more than 30% of stained cells, making the differential diagnosis with SMLPD particularly difficult without molecular data.In these patients, a monoclonal B-cell population was found, without monoclonal T-cell population, and the diagnosis of MZL could be made.The study of clonality may therefore be helpful in these cases of MZL with TFH hyperplasia of the microenvironnement 10 .Moreover, this study also highlights the value of comparing molecular techniques in cases of suspected cutaneous localization of systemic lymphoma.discovery of the same monoclonal population (AITL no 3) or same NGS pathogenic variants (AITL no 7, 9 and 10), in skin biopsy and lymph node can, indeed, allow to link two locations of the same disease. Taken individually, TNGS also identified pathogenic variants helpful for the diagnosis in 42% of MZL and 64% of cAITL.Even if these cases do not represent a large majority, they are not negligible considering the importance of an optimal classification of these lesions for the management and prognosis. In the present study, 47% of MZL presented pathogenic variants, involving TNFAIP3 (n = 6, 32%), KMT2D (n = 3, 16%), EP300 (n = 4, 21%), NOTCH2 (n = 3, 16%), and CARD11 (n = 2, 10.5%).According to the Cosmic database, NOTCH2 (19%), KMT2D (15%), and TNFAIP3 (11%) are the top 3 most frequent mutated genes observed in mucosa-associated lymphoid tissue (MALT) lymphomas 28 and nodal MZL 29 .EP300 and CARD11 are also frequently described in MZL 30 .FBW7 has not been considered as helpful 31 .Mutations do not seem correlated to prognosis in MZL: 44% of indolent cases among the mutated cases and 60% of relapses among the non-mutated cases.However, the panel targeted the most relevant genes for lymphoma diagnosis but did not cover all exons for some genes (e.g., KMT2D) and lacked a few select genes (e.g., FAS, SLAMF1, SPEN, and NCOR2), which are described in cutaneous MZL 10,32 .This could explain the negativity of some cases and the lack of correlation with the clinical course. Concerning AITL, most studies concern nodal locations, in which mutations of TET2 (52-76%), IDH2 (20-45%), DNMT3A (30-40%) and RHOA G17V (28-70%) seem frequent [11][12][13][14][15][16] .The only study currently published in cutaneous localizations is the one of Leclaire Alirkilicarslan et al., which included 41 patients and found IDH2 R172K/S and RHOA G17V mutations in 19% and 78% of cases respectively using PCR 17 .This study is the only one to study mutations using TNGS on a cohort of cutaneous localizations of AITL.Among them, 64% presented pathogenic variants involving TET2 (n = 7, 64%), RHOA (n = 4, 36%), and NOTCH1 (n = 1, 9%).Leclaire Alirkilicarslan et al. found 78% of RHOA G17V and 19% of cases with IDH2 R172 substitutions using ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare no competing interests. ", "section_name": "Competing interests", "section_num": null } ]
10.1186/s11658-019-0163-z	Non-contact co-culture with human vascular endothelial cells promotes epithelial-to-mesenchymal transition of cervical cancer SiHa cells by activating the NOTCH1/LOX/SNAIL pathway	The aim of this study was to investigate the effect of human umbilical vein endothelial cells on epithelial-to-mesenchymal transition of the cervical cancer cell line SiHa by studying the Notch1/lysyl oxidase (LOX)/SNAIL1 pathway.Monocultures of SiHa cells, SiHa cells containing a control sequence, and Notch1-silenced SiHa cells, as well as co-cultures of human umbilical vein endothelial cells with SiHa cells and Notch1-silenced SiHa cells, were established. The invasiveness of SiHa cells in each group was evaluated using a Transwell assay. The mRNA levels of E-cadherin and vimentin were detected using quantitative real-time polymerase chain reaction. The expression levels of the matrix metalloproteinases MMP-2 and MMP-9 were determined in SiHa cells using an immunofluorescence assay and the protein activity was detected by gelatin zymography. Changes in LOX, SNAIL1 and NOTCH1 expression in the SiHa cells in each group were detected using western blotting.Compared with monocultured SiHa cells, co-cultured SiHa cells showed a significant increase in their invasiveness and expression levels of vimentin, as well as of NOTCH 1, LOX, and SNAIL1, whereas their expression of E-cadherin was significantly reduced and protein activities of MMP-2 and MMP-9 were increased. Compared with SiHa, mono- and co-cultured NOTCH 1-silenced SiHa cells showed significant reductions in their invasiveness and expression levels of vimentin, NOTCH 1, LOX, and SNAIL1, whereas their expression of E-cadherin significantly increased and protein activities of MMP-2 and MMP-9 decreased.Co-culture with human umbilical vein endothelial cells promoted the epithelial-to-mesenchymal transition of SiHa cells by activating the NOTCH1/LOX/SNAIL1 pathway in SiHa cells, which enhanced their invasive and metastatic capacities. The results of this study may provide a new perspective on cervical cancer metastasis and a theoretical basis for clinical treatment.	[ { "section_content": "Cervical cancer is the fourth most common type of cancer in women worldwide, with an estimated 530,000 new cases each year.It is one of the leading causes of cancer-related deaths in women, with an estimated 270,000 deaths annually [1].Approximately 85% of cervical cancer deaths in the world occur in less developed or developing countries [2].In China, the incidence and rate of mortality from cervical cancer continue to rise; for example, in 2015 alone, the number of new cervical cancer cases reached 98,900, thus accounting for 18.7% of the global incidence and becoming a major public health issue in China [3].Studies have found that cervical cancer is more prone to metastasis than other types of cancer, and is one of the leading causes of death in patients with cervical cancer.Therefore, inhibiting or delaying metastasis of cervical cancer cells is of great significance in prolonging survival and improving the quality of life of patients.Cervical cancer metastasis is closely related to epithelial-to-mesenchymal transition (EMT), whereby epithelial tumour cells acquire a mesenchymal phenotype, which allows for the invasion and metastasis of tumour cells [4].Studies have found that abnormally elevated levels of multiple factors, including long noncoding RNAs, microRNAs, and transforming growth factor (TGF)-β, as well as signalling pathways such as the nuclear factor-κB, WNT, and NOTCH pathways, can activate EMT and promote metastasis in cervical cancer [5][6][7][8][9][10]. In tumour tissues, tumour and non-tumour cells interact to promote tumour development.Cancer progression is closely associated with the tumour microenvironment, including fibroblasts, immune cells, endothelial cells, blood vessels and proteins produced [11].Among non-tumour cells, inflammatory cells are implicated in the persistent proliferation and immunosuppression-mediated escape of tumour cells [12].In addition, the hypoxia-induced migration of endothelial cells and angiogenesis play important roles in promoting tumour growth, metastasis, and progression [13].EMT confers characteristics of mesenchymal cells to tumour cells, which then exhibit high motility and can easily enter the bloodstream by degrading tumour tissues and blood vessel walls, resulting in metastasis.Non-contact culture can make tumour cells interact with other cells through paracrine factors, providing a microenvironment for tumour cells, and allowing further studies of the formation, occurrence and development of cancer, as well as the treatment mechanism [11]. Interactions between tumour cells and vascular endothelial cells have been shown to promote tumour cell metastasis in multiple tumour types [14,15].Previous studies have mainly focused on the effects of tumours on blood vessel formation [16].However, considering cell-cell interactions and the bidirectionality of signal transduction, it is necessary to evaluate whether vascular endothelial cells can induce EMT of cervical cancer cells and promote tumour cell metastasis.Thus, in this study, we utilised noncontact co-culture of human vascular endothelial cells and the cervical cancer cell line SiHa to investigate the potential role and molecular mechanisms of normal human vascular endothelial cells in cervical cancer metastasis. ", "section_name": "Background", "section_num": null }, { "section_content": "", "section_name": "Methods", "section_num": null }, { "section_content": "HEK293 cells and the human cervical cancer cell line SiHa were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences.Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection.Two recombinant adenoviruses, Ad-control, with an empty capsid containing a control sequence, and Ad-Not-siRNA, containing a gene encoding a NOTCH 1-specific small interfering RNA (siRNA), were constructed by Sangon Biotech Co., Ltd.(Shanghai, China).High-glucose Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco, Thermo Fisher Scientific (Waltham, MA, USA).Foetal bovine serum (FBS) was purchased from Beijing Ever Green Biotechnology Co., Ltd.Trypsin, RIPA lysis buffer, hypersensitive enhanced chemiluminescence (ECL) detection reagents, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) reagents, and western blotting membranes were purchased from Beyotime Biotechnology Co., Ltd.(Shanghai, China).Rabbit monoclonal antibodies against matrix metalloproteinase (MMP)-2, MMP-9, NOTCH 1, and SNAIL1 were purchased from Abcam (Cambridge, UK).DyLight 594-labelled goat anti-rabbit IgG was purchased from GeneTex (Irvine, CA, USA).Horseradish peroxidase-labelled goat anti-rabbit IgG was purchased from Cell Signaling Technology.Trizol, a reverse transcription kit, and real-time quantitative PCR kit were purchased from TaKaRa (Tokyo, Japan).Primers were synthesised by TaKaRa, and the primer sequences are shown in Table 1. ", "section_name": "Cells and reagents", "section_num": null }, { "section_content": "Both SiHa cells and HUVECs were cultured in high-glucose DMEM containing 10% FBS at 37 °C, in an atmosphere of 5% CO 2 .Cells were passaged when they reached 80-90% confluence. ", "section_name": "Cell culture", "section_num": null }, { "section_content": "For virus propagation, 1 μl of Ad-control or Ad-Not-siRNA viral stock solution was added to SiHa cells in the logarithmic growth phase.When the cells appeared rounded and bead-like and approximately 60% were afloat, they were collected by centrifugation, frozen in liquid nitrogen for 15 min, then thawed in a 37 °C water bath, and vortexed for 1 min.The freeze-thaw process was repeated three times, after which the cells were centrifuged at 11,950 g for 5 min at 4 °C.The virus-containing supernatants were collected and added to HEK293 cells for repeated infection.The recombinant adenoviruses were repeatedly propagated using the above-described procedure until high titres were obtained. HEK293 cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well.After 8 h of cell incubation, the obtained viral stock solutions were diluted to 1:10 2 -1:10 5 and 100 μl was added to cells in triplicate wells.The number of viruses was counted after 24 h, and the virus titre was calculated according to the following formula: virus titre = Table 1 Primer sequences number of positive cells × virus dilution factor / 0.1.The final recombinant adenovirus titres were 5 × 10 10 plaque-forming units per microliter.Healthy growing SiHa cells were seeded in 24-well plates and after reaching 50% confluence were infected with 0.1, 0.3, 0.5, 0.7, 0.9, or 1.1 μl of the recombinant adenoviruses in quadruplicate at the multiplicity of infection (MOI) of 5, 15, 25, 35, 45, and 55, respectively.The cells were observed at 24, 48, and 72 h, separately, and the fluorescent signal and cell growth status were recorded.The titre corresponding to an infection rate of > 70%, without affecting the cell conditions (MOI, 25), was selected as the optimal infectious titre.The remaining viruses were aliquoted and stored at -80 °C until use. ", "section_name": "Propagation of recombinant adenoviruses and selection of optimal infectious titres", "section_num": null }, { "section_content": "SiHa cells were sub-cultured and after reaching 50-60% confluence were infected with the Ad-control and Ad-Not-siRNA adenoviruses at the optimal infectious titres.The fluorescence intensity in each group of cells was recorded after 24 h.Uninfected SiHa cells were used as a blank control group. ", "section_name": "Recombinant adenovirus infection", "section_num": null }, { "section_content": "SiHa cells were cultured as the following five groups (each in triplicate): monocultures of SiHa cells (SiHa group), monoculture of SiHa cells containing control sequence (Adcontrol group), monoculture of NOTCH 1-silenced SiHa cells (Ad-Not-siRNA group), co-culture of HUVEC/SiHa cells (HUVEC/SiHa group), and co-culture of HUVEC/ NOTCH 1-silenced SiHa cells (HUVEC/Ad-Not-SiHa group).SiHa cells and NOTCH 1-silenced SiHa cells in the logarithmic growth phase were seeded in 6-well plates at a concentration of 1 × 10 6 /ml in a total volume of 2.5 ml/well.In Transwell co-culture systems, 1.5 ml of HUVECs at a concentration of 1 × 10 5 /ml was added to the upper chamber.High-glucose DMEM containing 10% FBS was used as the medium for both monocultures and co-cultures.The SiHa cells in the lower chambers were collected after 48 h of incubation in all groups. ", "section_name": "Establishment of co-culture systems", "section_num": null }, { "section_content": "SiHa cells and NOTCH 1-silenced SiHa cells in the logarithmic growth phase were seeded at a concentration of 1 × 10 5 /ml in a total volume of 1.5 ml onto the Matrigel matrix coating the upper chambers of the Transwell systems.In the co-culture systems, 2.5 ml of HUVECs at a concentration of 1 × 10 5 /ml were added to the lower chambers.High-glucose DMEM containing 10% FBS was used as the medium for both the monocultures and the co-cultures.After 48 h of culture incubation, the Transwell chambers were removed and fixed in anhydrous methanol at -20 °C, followed by washing with phosphate-buffered saline (PBS).Non-invading cells were gently wiped off with cotton swabs.The chambers were then stained with crystal violet for 3 min.After the wells were washed with PBS, five fields of view were randomly selected for each well, and the invaded cells were photographed and counted. ", "section_name": "Cell invasion assay", "section_num": null }, { "section_content": "SiHa cells from each group were collected after 48 h of incubation, and 1.5 ml of TRIzol reagent was added on ice.The cells were allowed to stand at room temperature for 5 min, followed by centrifugation at 13,000 rpm for 5 min.The supernatants were mixed with chloroform, followed by centrifugation and precipitation with isopropanol.After centrifugation, the DNA precipitates were washed with 75% ethanol, then dried and dissolved in diethyl pyrocarbonate treated water.Genomic DNA removal, reverse transcription, and qPCR amplification were performed according to the instructions for the TaKaRa kit.The amplification conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C, 15 s, 60 °C, 15 s, and 72 °C, 30 s. mRNA levels of the target genes were calculated using the 2 -ΔΔCt method. ", "section_name": "Quantitative real-time PCR", "section_num": null }, { "section_content": "Gelatine zymography was used to semi-quantitatively determine protein and activity levels of MMP-2 and MMP-9.Briefly, proteins were separated by SDS-PAGE in gels containing 1 mg/ml gelatine.The gels were then treated with 2.5% Triton X-100 for 30 min at room temperature.The zymograms were subsequently incubated overnight at 37 °C in developing buffer.The gels were stained with 0.5% Coomassie blue R-250 and destained in 10% acetic acid and 40% ethanol in dH 2 O. Image acquisition software (UVP Inc., USA) was used for densitometric analysis of lytic bands. ", "section_name": "Gelatine zymography", "section_num": null }, { "section_content": "SiHa cells were washed with PBS in 6-well plates and fixed with 4% paraformaldehyde for 15 min at room temperature, followed by washing with PBS.Next, 0.25% Triton X-100 was added for 15 min for membrane permeabilisation, followed by incubation with a 5% bovine serum albumin blocking solution containing 0.25% Triton X-100 for 30 min.After blocking, the cells were incubated with primary MMP-2 and MMP-9 (diluted 1:500 in the blocking solution) overnight at 4 °C, followed by washing with PBS and incubation for 1 h at room temperature with DyLight 594-labelled secondary antibodies (diluted 1:2000).Finally, the cells were washed with PBS and photographed under a fluorescence microscope.The Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) was used to analyse the fluorescence. ", "section_name": "Immunofluorescence assays", "section_num": null }, { "section_content": "SiHa cells from each group were washed with PBS and incubated with RIPA lysis buffer on ice.The lysates were centrifuged at 13,000 rpm, and protein concentrations were determined in the supernatants.The proteins were denatured by boiling in 4× loading buffer for 5 min and stored at -20 °C until use.Equal amounts of protein were separated by electrophoresis in 10% separation gel and 5% stacking gel and then transferred to membrane, which were blocked and incubated overnight with primary antibodies against NOTCH 1, LOX, -SNAIL1, and β-actin (all diluted 1:800).The membranes were then washed with PBS, incubated with secondary antibodies (diluted 1:2000) for 1.5 h at room temperature, washed with PBS again, and visualised with ECL reagents.X-ray films were developed and photographed.The Image-Pro Plus software was used to analyse the density of the immunoreactive bands. ", "section_name": "Western blotting", "section_num": null }, { "section_content": "Data were analysed using the SPSS 21.0 software (IBM, Armonk, NY, USA).Multivariate analysis of variance was used for comparisons among multiple groups and the least significant difference t test was used for comparisons between two groups.Differences with P < 0.05 were considered statistically significant. ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "The results showed that, compared with the SiHa group, there was no significant difference in Notch1 levels in SiHa cells in the Ad-control group, whereas a significant increase in Notch1 levels was observed in the SiHa cells in the HUVEC/SiHa group.Compared with the Ad-control group, the Notch1 levels were significantly lower in the Ad-Not-siRNA and HUVEC/Ad-Not-SiHa groups (Fig. 1a,b). ", "section_name": "NOTCH 1 expression in SiHa cells", "section_num": null }, { "section_content": "The cell invasion assay showed that there was no significant difference in the invasiveness between SiHa cells without and with the Ad-control, whereas SiHa cells from the HUVEC/SiHa group showed a significantly higher invasive capacity and displayed a more mesenchymal morphology, with a spindle-like shape.Compared with that in the Ad-control group, the invasive capacity was significantly lower in SiHa cells from the Ad-Not-siRNA and HUVEC/Ad-Not-SiHa groups, both of which exhibited a more cobblestone-like cell phenotype (Fig. 2a,b). ", "section_name": "SiHa cell invasiveness", "section_num": null }, { "section_content": "qPCR showed that there were no significant differences in the mRNA levels of Ecadherin and vimentin between SiHa cells without and with the Ad-control, whereas in SiHa cells from the HUVEC/SiHa group, the mRNA level of vimentin was significantly increased, while that of E-cadherin was significantly decreased.Compared with those from the Ad-control group, SiHa cells from the Ad-Not-siRNA and HUVEC/Ad-Not-SiHa groups were showed a significant decrease in the protein mRNA levels of vimentin and a significant increase in the mRNA levels of E-cadherin (Fig. 3).demonstrated to stabilise SNAIL1.Therefore, we evaluated the levels of LOX and SNAIL1 proteins using western blotting.The results showed that there were no significant differences in the protein levels of LOX and SNAIL1 between SiHa cells without and with the Ad-control, whereas levels were significantly increased in SiHa cells from the HUVEC/SiHa group.Compared with those in the Ad-control group, the LOX and SNAIL1 protein levels were significantly decreased in SiHa cells from the Ad-Not-siRNA and HUVEC/Ad-Not-SiHa groups (Fig. 4a,b). ", "section_name": "E-cadherin and vimentin mRNA levels in SiHa cells", "section_num": null }, { "section_content": "", "section_name": "LOX and SNAIL1 protein expression in SiHa cells", "section_num": null }, { "section_content": "", "section_name": "SNAIL1 has been previously shown to inhibit E-cadherin expression and activate vimentin expression during EMT, thereby promoting EMT. In addition, LOX has been", "section_num": null }, { "section_content": "Gelatin zymography was used to detect pro-and active forms of MMP-2 and MMP-9.Both MMP-2 and MMP-9 were expressed in all samples, and the differences between SiHa cells without and with the Ad-control were not significant.In the HUVEC/SiHa, the MMPs activities were significantly increased, and in the Ad-Not-siRNA and HUVEC/Ad-Not-siRNA groups, they were significantly reduced (Fig. 5a-d). ", "section_name": "MMP-2 and MMP-9 protein expression", "section_num": null }, { "section_content": "In epithelial cell-derived tumours, EMT is critical for them to acquire characteristics such as a reduced level of differentiation and increased metastatic potential.EMT is an important driver of tumour progression, and the process may be promoted by many factors.EMT is regulated at multiple levels by a regulatory network that includes tissues, cells, molecules, and the environment.At the cellular level, interactions between tumour and non-tumour cells play an important role in regulating EMT of the former.An example of such non-tumour cells is the vascular endothelial cells, which are single-layered squamous cells that line the inner surfaces of blood vessels, lymphatic vessels, and the heart, among other structures, forming the inner layer of the blood vessel wall.In tumour tissues, vascular endothelial cells interact with tumour cells to promote their EMT.Studies have found that vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF) secreted by vascular endothelial cells in head and neck squamous cell carcinoma promote EMT and metastasis of tumour cells via the VEGFR-2/HEF1/AKT/Twist1 pathway.In the present study, we found that, compared with that of SiHa cells in monoculture, the invasion capacity of SiHa cells co-cultured with HUVECs was significantly increased, suggesting that HUVECs promote the metastasis of SiHa cells.Moreover, we found that HUVECs could increase the expression of vimentin and SNAIL1 and inhibit that of E-cadherin in SiHa cells.During EMT, there is a loss of epithelial markers such as E-cadherin and cytokeratin, resulting in the loss of cell polarity and intercellular junctions.Furthermore, upregulation of mesenchymal markers, such as MMPs, vimentin, and α-smooth muscle actin, induces morphological changes in cells and enhances their ability to degrade the extracellular matrix.Overall changes in these factors cause cells to undergo EMT and promote tumour metastasis [17].The transcription factor Snail can regulate EMT; in particular, activated Snail recognises and binds to the E-box sequence of the E-cadherin gene, which inhibits its expression and promotes the EMT of cells [18,19].In summary, HUVECs may promote EMT and induce metastasis of SiHa cells. EMT may be affected by the activity of many signalling pathways.Multiple studies have found that the Notch signalling pathway is closely associated with EMT in cervical cancer.Zagouras et al. [20] showed that Notch1 was expressed in carcinoma in situ and in invasive squamous cell carcinoma of the cervix.In addition, a higher level of the NOTCH 1 receptor was detected in cervical adenocarcinoma tissues, whereas it was absent in normal cervical tissues, implicating NOTCH 1 receptors in cervical cancer.Daniel et al. [21] found that Notch1 was activated during the progression of grade III cervical intraepithelial neoplasia to cervical cancer, with intense staining of NOTCH 1 in both the cytoplasm and the nucleus.The expression of NOTCH 1 receptor gradually increased during the progression from cervical intraepithelial neoplasia to squamous cell carcinoma of the cervix, demonstrating that NOTCH 1 was highly expressed in the tissues of cervical cancer.The results of the previous study also showed that, during progression from grade III cervical intraepithelial neoplasia to micro-invasive carcinoma, the localisation of NOTCH 1 receptor shifted from the cytoplasm to the nucleus.In vitro and in vivo studies have shown that the introduction of an antisense oligonucleotide to human NOTCH 1 into the HPV16-positive cervical cancer cell line Ca Ski can inhibit tumour cell growth and reduce the tumourigenicity of the NOTCH 1 receptor.These results indicate that NOTCH 1 is essential in the transformation of cervical epithelial cells.In cervical cancer, TGF-β can activate the NOTCH 1 receptor, induce Snail expression, inhibit E-cadherin expression, and promote EMT.We thus assessed whether HUVECs could induce the metastasis of SiHa cells through NOTCH 1 and found that silencing of NOTCH 1 expression in SiHa cells significantly decreased the invasive capacity.In non-contact co-cultures of NOTCH 1-silenced SiHa cells and HUVECs, the promoting effect of HUVECs on the invasiveness of SiHa cells was lost, suggesting that HUVECs induced metastasis of SiHa cells through Notch1.Further molecular studies showed that following NOTCH 1 knockdown, the HUVEC-induced increase in the expression of vimentin and SNAIL1 and the decrease in the expression of E-cadherin were abolished.These results demonstrate that HUVECs can promote EMT and induce metastasis of SiHa cells by activating NOTCH 1. ", "section_name": "Discussion", "section_num": null }, { "section_content": "In summary, we found that HUVECs promote metastasis of the cervical cancer cell line SiHa, which may potentially be attributed to a HUVEC-secreted protein that acts on NOTCH 1 in SiHa cells, which in turn activates the EMT in the SiHa cells.The putative protein remains to be identified in future research. ", "section_name": "Conclusions", "section_num": null } ]	[ { "section_content": "This work was supported by grants from the National Natural Science Foundation of China (No. 81201549), Gansu Provincial People's Hospital fund (18GSSY5-15). ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "National Natural Science Foundation of China (No. 81201549), Gansu Provincial People's Hospital fund (18GSSY5-15). ", "section_name": "Funding", "section_num": null }, { "section_content": "The data that support the findings of this study are available from the corresponding author upon reasonable request. ", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "Ethics approval and consent to participate Not applicable. Not applicable. The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Consent for publication", "section_num": null }, { "section_content": "Ethics approval and consent to participate Not applicable. ", "section_name": "", "section_num": "" }, { "section_content": "Not applicable. ", "section_name": "Consent for publication", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.2174/157340308785160552	Notch Signaling in Cardiovascular Disease and Calcification	Recent increase in human lifespan has shifted the spectrum of aging-related disorders to an unprecedented upsurge in cardiovascular diseases, especially calcific aortic valve stenosis, which has an 80% risk of progression to heart failure and death. A current therapeutic option for calcified valves is surgical replacement, which provides only temporary relief. Recent progress in cardiovascular research has suggested that arterial and valve calcification are the result of an active process of osteogenic differentiation, induced by a pro-atherogenic inflammatory response. At molecular level, the calcification process is regulated by a network of signaling pathways, including Notch, Wnt and TGFbeta/BMP pathways, which control the master regulator of osteogenesis Cbfa1/Runx2. Genetic and in vitro studies have implicated Notch signaling in the regulation of macrophage activation and cardiovascular calcification. Individuals with inactivating Notch1 mutations have a high rate of cardiovascular disorders, including valve stenosis and calcification. This article reviews recent progress in the mechanism of cardiovascular calcification and discusses potential molecular mechanisms involved, focusing on Notch receptors. We propose a calcification model where extreme increases in vascular wall cell density due to inflammation-induced cell proliferation can trigger an osteogenic differentiation program mediated by Notch receptors.	[ { "section_content": "E n e r g y S c i e n c e , E n g i n e e r i n g , a n d T e c h n o l o g y R e c e n t P a t e n t s o n E n g i n e e r i n g Cu r r e n t A l t e r n a t i v e E n e r g y R e c e n t P a t e n t s o n Me c h a n i c a l E n g i n e e r i n g E n g i n e e r i n g R e c e n t P a t e n t s o n E n g i n e e r i n g I n t e r n a t i o n a l J o u r n a l o f S e n s o r s , I n t e r n a t i o n a l J o u r n a l o f S e n s o r s , Wi r e l e s s Co mmu n i c a t i o n s a n d Co n t r o l 2 4 0 0 2 0 0 0 2 1 6 0 2 8 0 0 1 7 6 0 1 7 6 0 9 6 ", "section_name": "", "section_num": "" } ]	[]
10.1186/s13046-022-02294-5	HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy	<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on <jats:italic>FCGR2B</jats:italic> gene transcription.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>We report that TAMs are FcγRIIb<jats:sup>bright</jats:sup> relative to healthy tissue counterparts and under hypoxic conditions<jats:italic>,</jats:italic> mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb<jats:sup>+<jats:italic>/</jats:italic>+</jats:sup> transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.</jats:p> </jats:sec>	[ { "section_content": "Hypoxia is a state that arises when cellular demand for molecular oxygen (O 2 ) exceeds supply [1].Several studies have reported that hypoxia is a distinctive aspect of a wide range of solid tumors [2][3][4][5][6][7][8][9] and over half of tumor regions exhibit lower O 2 levels relative to their healthy tissue counterparts [10].In the atmosphere, pO 2 is 160 mmHg (21.1%), falling to 100 mmHg (13.2%) in arterial blood [11].In comparison, in pancreatic ductal adenocarcinoma, median pO 2 is 0-5.3 mmHg (0-0.7%)compared to 24.3-92.7 mmHg (3.2-12.3%) in donor matched healthy pancreas [5].Cells respond to hypoxia by stabilizing the hypoxia-inducible factor (HIF) family of transcription factors.In the tumor microenvironment (TME) the genes induced by HIF-1α and HIF-2α enhance tumor growth and survival, by increasing angiogenesis, cell survival, cell proliferation, metastasis, pH regulation, glycolysis and maintenance of cancer stem cells [12]. Among the diverse cell populations present in the TME, macrophages are often the most abundant and are referred to as tumor-associated macrophages (TAMs) [13].Macrophages exist in multiple states of activation with so-called M1 and M2 describing their extremes; M1 macrophages (generated through LPS/IFN-γ stimulation) are pro-inflammatory and are thought to possess antitumor functions; M2 macrophages (produced following Interleukin (IL)-4/IL-13 treatment) are considered antiinflammatory and pro-tumor [14,15].Although TAMs are thought to acquire a primarily proangiogenic tumor promoting (M2-like) phenotype in the TME [16]. Clinically important tumor targeting monoclonal antibodies (mAb) such as Rituximab, Herceptin and Cetuximab, function, at least in part, by inducing mononuclear phagocytes to deplete tumor cells [18][19][20][21][22][23].Furthermore, mAbs such as Ipilimumab, targeting immune checkpoint molecules, previously thought to function solely via receptor blockade and expansion of effector T (Teff ) cells [24], have also recently been reported to work optimally through myeloid-cell mediated depletion of tumor infiltrating immunosuppressive regulatory T (Treg) cells [25][26][27]. A key mechanism by which direct targeting anti-cancer mAbs deplete cellular targets in the TME is via antibody dependent cellular phagocytosis (ADCP) which is primarily accomplished by macrophages [28].As such, mAb-bound target cells interact with the activating Fc gamma receptors (FcγRs); FcγRI, FcγRIIa and FcγRIIIa for optimal ADCP (FcγRI, FcγRIII and FcγRIV in the mouse), whereas engagement with the sole inhibitory FcγR, FcγRIIb (FcγRII in mice) attenuates phagocytic function [29].Expression levels and cellular distribution of FcγR on effector cells are therefore of crucial importance in antibody therapy outcome. Although an important feature of many tumors, the impact of physiological hypoxia on anti-cancer mAb immunotherapy has not been investigated in detail to date.In the current study we applied a multi-OMIC approach to profile the effects of hypoxia on FcγR expression in mononuclear phagocytes and its subsequent impact on antitumor mAb effector functions.We demonstrate that exposure to physiological or pharmaceutical hypoxia, induces transcriptionally driven and rapid upregulation of FcγRIIb expression on mononuclear phagocytes.Hypoxia-mediated enhancement of FcγRIIb expression impairs ADCP and reduces in vivo therapeutic mAb efficacy in murine tumor models.We provide a detailed molecular and cellular basis for tumor hypoxia driven resistance to mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME that requires evaluation for current and novel mAb immunotherapies to improve clinical efficacy.cell carcinoma (RCC) and non-cancerous healthy kidney tissue samples were obtained from resected kidneys from 5 RCC patients (REC reference number: 17/WA/0241).Lymphocele samples (30-100 mL) were sourced from 3 anonymized breast cancer patents (REC reference number: 10/H0504/73, for breast cancer patient samples).Peripheral blood samples were taken from Chronic lymphocytic leukemia (CLL) patients, PBMCs were isolated and placed in 90% Fetal calf serum (FCS)/10% Dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored in liquid nitrogen until further use as target cells in monocytederived macrophage (MDM) based phagocytosis assays (REC reference number: 10/H0504/187, for CLL patient samples).These aforementioned clinical samples were released from the Human Tissue Authority Licensed University of Southampton, Cancer Sciences Tissue Bank, as approved by the Southampton and South West Hampshire Research Ethics Committee (REC reference: 280/99).All informed consent for the use of human material was provided in accordance with the Declaration of Helsinki. ", "section_name": "Background", "section_num": null }, { "section_content": "Mice were used in these studies as the least sentient species with an immune system comparable to humans.The availability of a transgenic (Tg) mouse strain expressing human (h) FcγRIIb also facilitates more detailed understanding of the effects of hypoxia on mAb mediated cell target depletion in a living organism to inform clinical translation.Wild type (WT) C57BL/6 and hFcγRIIB +/-x mouse (m) FcγRII -/-x hCD20 +/-C57BL/6 J mice were described previously [30] and were maintained and bred in house.Splenocytes from hCD20 +/-x mFcγRII -/-were used as target cells in the adoptive transfer in vivo experiment in Fig. 7i-j (NB: mFcγRII -/-cells were selected to remove any potential influence from mFcγRII changes on the target cells).Genotypes were confirmed by PCR and/or flow cytometry.All mice were bred in a closed research facility under specific pathogen-free conditions in individually ventilated cages (IVCs).Following approval by local ethical committees, reporting to the Home Office Animal Welfare Ethical Review Board (AWERB) at the University of Southampton, in vivo experiments were conducted under UK Home Office Project licenses P81E129B7 and P4D9C89EA.Experiments used both male and female mice, and mice were age and sex matched within experiments.For the majority of experiments mice were aged between 8-15 weeks.Littermates of the same sex were randomly assigned to experimental groups at the start of the experiment.Mice were maintained on a 12-h light/dark cycle, food and water was made available at all times, environmental enrichment was provided, and temperature was maintained between 20-24 °C.Mice were visually checked daily if adverse effects were anticipated or if mice were nearing a humane end-point. ", "section_name": "Mice", "section_num": null }, { "section_content": "To prepare myeloid cells from murine spleens for flow cytometric analysis, harvested tissue was cut into small pieces, placed in 5 mL complete RPMI (RPMI-1640 supplemented with, 2 mM L-glutamine, 1 mM pyruvate, 100 U/mL penicillin, 100 mg/mL streptomycin and 10% heat inactivated HyClone FCS (GIBCO)) and mechanically dissociated via feeding the tissue through a 70 μm BD Falcon cell filter (BD Biosciences) to achieve a single cell suspension. 1 mL of ammonium chloride buffer (154.4 mM ammonium chloride and 10 mM potassium bicarbonate (Sigma-Aldrich)) was added to lyse red blood cells (RBCs) in each spleen sample and samples were washed once in complete RPMI.To isolate immune cells from the peritoneum, mice were sacrificed, and 5 mL of ice-cold phosphate buffered saline (PBS) was injected into the peritoneum using a 10 mL syringe and a 25-G needle.The peritoneum was then gently massaged, and the PBS drawn back into the syringe to achieve a single cell suspension.To prepare bone marrow immune cells for flow cytometric analysis, mice were sacrificed and hind femora and tibiae isolated.Muscle and soft tissue were removed from the bones and each bone trimmed at both ends.Bone marrow was then flushed with complete RPMI until bones were white.The liberated cells were passed through a 70 μm BD Falcon cell filter (BD Biosciences) to achieve a single cell suspension.All murine livers and tumors were cut into small 2 mm × 2 mm pieces, incubated in 1.5 mL serum free RPMI 1640 per liver or 500 mm 3 tumor, with 200 U/mL DNase I (Sigma-Aldrich) and 15 U/mL Liberase TL (Roche Diagnostics) for 30 min, at 37 °C in a shaking incubator.25 mL complete RPMI was added per sample and the digested samples were mechanically dissociated and, together with the cell suspension, passed through a 70 μm BD Falcon cell filter and centrifuged (300 × g for 5 min).Additionally, immune cells in liver samples were separated from nonimmune cells via Percoll (Sigma-Aldrich) density centrifugation [31].The supernatants from each tumor and liver sample were removed and 5 mL of ammonium chloride buffer added to lyse RBCs.Post-isolation, all murine tumor, liver, splenocyte, peritoneal lavage and bone marrow single cell suspensions were centrifuged (300 × g for 5 min) and resuspended in complete RPMI at 1 × 10 7 cells/mL, prior to staining with fluorophore conjugated antibodies. ", "section_name": "Isolation of murine immune cells", "section_num": null }, { "section_content": "PBMCs were isolated from leukocyte cones or from whole peripheral blood from mesothelioma patients, within 2 h of collection, by density gradient centrifugation at 800 × g for 20 min (Lymphoprep, Axis-Shield).Primary human monocytes were isolated using the Pan Monocyte Isolation Kit, human (Miltenyi Biotech) from 1 × 10 8 PBMCs per isolation, according to the manufacturer's protocol. Pleural fluid and lymphocele clinical samples were centrifuged at 300 × g for 10 min and the supernatants were removed.RBCs were lysed with Erythrolyse Red Blood cell (RBC) lysis buffer (AbD SeroTec) and samples washed once in PBS/1% Bovine Serum Albumin + 10% FCS (Sigma-Aldrich) and stained immediately with fluorophore conjugated mAbs for flow cytometric analysis. RCC or healthy (normal) kidney tissue (0.9-1.3 g of tissue per sample) was cut into small 2 mm × 2 mm pieces, incubated in 1.5 mL RPMI 1640 per 0.3 g of tissue, with 200 U/mL DNase I (Sigma-Aldrich) and 15 U/mL Liberase TM (Roche Diagnostics) for 45 min, at 37 °C in a shaking incubator.25 mL of complete RPMI was added per 0.3 g of tissue and the digested samples mechanically dissociated and, together with the cell suspension, passed through a 70 μm BD Falcon cell filter (BD Biosciences) and centrifuged (300 × g for 5 min).The supernatant was removed and 5 mL of ammonium chloride buffer added to lyse RBCs.Each sample was then centrifuged (300 × g for 5 min), the supernatant discarded, the cell pellet resuspended in complete RPMI with 10% Human AB serum (Invitrogen) at 1 × 10 7 cells/mL and incubated at room temperature for 15 min.These cell suspensions were then centrifuged (300 × g for 5 min), the supernatants discarded and resuspended in complete RPMI and stained immediately with fluorophore conjugated mAbs for flow cytometric analysis. ", "section_name": "Isolation of human immune cells", "section_num": null }, { "section_content": "Chinese hamster ovary (CHO) K1 (ATCC CCL-61) cells were cultured in complete RPMI 1640 with 0.05 mM β-mercaptoethanol (2ME; Sigma-Aldrich) and incubated at 37 °C, 5% CO 2 .CHO-K1 cells were then transfected with FcγRIIb1 or FcγRIIb2 isoforms in plasmid pcDNA3 [32], selected by using 1 mg/mL geneticin (Life Technologies), and screened by flow cytometry using the pan-FcγRII mAb AT10 F(ab') 2 -FITC (in-house).Positive colonies were expanded and then sorted using a FAC-SAria II flow cytometer (BD Biosciences).THP-1 (human leukemia monocytic cell line; ATCC TIB-202) cells were obtained from LGC Standards (Middlesex, UK).THP-1 cells were cultured in complete RPMI 1640 medium with 0.05 mM 2-ME, maintained at 0.25-0.5•10 6cells/ml and incubated at 37 °C, 5% CO 2 .THP-1 cells were passaged before reaching 1 × 10 6 cells/ml.EL4 cells (ATCC ® TIB-39 ™ ), a murine thymoma cell line, were cultured in complete RPMI 1640 supplemented with 0.05 mM 2-ME.Cells were maintained between 1 × 10 5 and 1 × 10 6 cells/mL and incubated at 37 °C, 5% CO 2 .These EL4 cells were then transfected with human CD20 in plasmid pcDNA3 [33], selected with 10 μg/mL puromycin (GIBCO).Human CD20 expression was screened by flow cytometry, using Rituximab (Roche) conjugated in-house using an Alexa Fluor ™ 488 Protein Labelling Kit (Ther-moFisher Scientific).Positive colonies were expanded and then sorted using a FACSAria II flow cytometer (BD Biosciences).MC38 (murine colon adenocarcinoma cell line, kindly gifted by Dr Sjef Verbeek), MCA205 (fibrosarcoma cell line, Sigma-Aldrich) and E.G7-OVA (T cell lymphoma cell line expressing model antigen hen egg ovalbumin, ATCC ® , CRL-2113 ™ ) were maintained in complete RPMI supplemented with 0.05 mM 2-ME.E0771 (breast carcinoma cell line, ATCC ® , CRL-3461 ™ ) cells were maintained in complete RPMI alone. ", "section_name": "Cell lines and transfections", "section_num": null }, { "section_content": "In the human monocyte phagocytosis assays, Rhesus D antigen positive RBCs were used as target cells via opsonization with wild type human IgG1 anti-Rhesus D mAb, kind-gift from Dr Gestur Vidarsson (Sanquin Blood Supply Foundation, Amsterdam, Netherlands).In Fig. 7c, to block IgG Fc-FcγRII interactions, E08 (antihuman-FcγRIIa) or 6G11 (anti-FcγRIIb), described previously [30,34], provided by BioInvent International AB (Lund, Sweden), were used as F(ab') 2 fragments to pretreat primary human monocytes, before assessment of RBC ADCP capacity.F(ab') 2 fragments were produced by trypsin digestion as before [35].mAb was purified using Protein A-Sepharose, and aggregates were removed by gel filtration.Preparations were endotoxin low (< 1 ng/ mg protein, Endosafe-PTS, Charles River Laboratories).For the human MDM ADCP assays, and when targeting human CD20 + target cells in vivo, clinical grade Rituximab (Roche) and Obinutuzumab (Roche) were used (kindly gifted by Prof Christian Klein, Roche).Cetuximab (Merck KGaA) and Herceptin (Roche, kindly gifted by Thomas Valerius) were used as isotype controls for the aforementioned anti-CD20 mAbs.For systemic B cell depletion in wild type C57BL/6 J mice in Additional file 7: Fig. S7h, anti-murine CD20 antibody, clone 18B12 (produced in-house) was used as a mouse IgG2a to systemically deplete B cells as previously described [36,37]. ", "section_name": "Antibodies", "section_num": null }, { "section_content": "PBMCs were isolated from leukocyte cones from healthy donors and were seeded 2 × 10 7 cells per well in 6-well plates (Corning Centristar), in complete RPMI (supplemented with 1% human AB serum, Invitrogen) at 37 °C and 5% CO 2 for 2 h.Non-adherent cells were removed with PBS and cells cultured overnight in complete RPMI.Adherent monocytes were then differentiated with M-CSF (100 ng/ml) on days 2, 4 and 6 with or without Dimethyloxalylglycine (DMOG) or Roxadustat (both at 20 µM).On Day 7 post-culture, MDMs were left untreated or stimulated with IFN-γ (50 ng/mL) and LPS (2 ng/mL), or IL-4 (20 ng/mL) and IL-13 (10 ng/mL), to generate M0, M1 and M2-like macrophages, respectively.Changes in cell morphology were assessed by phase contrast microscopy (Axiovert 135, Zeiss).Phenotypic and functional characterization of MDMs was performed 9 days post-culture: MDMs were removed from the plates with a cell scraper, 2 × 10 5 cells were transferred to each FACS tube and analyzed using BD FACSCantoII or FACSCalibur (BD Biosciences) flow cytometers and FlowJo Version 10 software (FlowJo LLC). ", "section_name": "Generation of monocyte-derived macrophages (MDMs)", "section_num": null }, { "section_content": "In Fig. 1, PBMCs from heathy human subjects were cultured at low density (LD) defined as 1 × 10 6 cells/mL or at high density (HD) defined as 1 × 10 7 cells/mL, in serum free complete CTL-Test Medium ((Europe GmbH, Bonn, Germany), supplemented with glutamine (2 mM), pyruvate (1 mM), penicillin (100 IU/mL), and streptomycin (100 IU/ mL)), at 37 °C in 5% CO 2 .1.5 mL of cells at the aforementioned concentrations were cultured in flat bottomed 24-well plates (Corning Centristar) for 48 h before immunophenotyping using flow cytometry and Western blotting. To determine whether physiological hypoxia leads to FcγRIIb upregulation on human monocytes, LD PBMCs or purified monocytes were incubated in a hypoxic chamber (Billups-Rothenberg, Inc).Cells were cultured in complete CTL medium in a 24-well plate and placed in the hypoxic chamber.To create hypoxic conditions, the chamber was attached to a gas cylinder containing 1% O 2 , 5% CO 2 and 94% N 2 with tubing using a flow meter incorporated in a regulator.The chamber was gassed at a flow rate of 20 L/minute for 7-10 min and then sealed whilst being filled with 1% O 2 .This step was repeated an hour later.Cells in the sealed hypoxic chamber were then kept inside a conventional incubator at 37 °C for 48 h before FcγR expression levels were assessed using flow cytometry.PBMCs or purified monocytes were also cultured at 3% O 2 levels in complete CTL medium, in a Thermo Scientific tri-gas incubator (Thermo Fisher Scientific), at 37 °C and 5% CO 2, followed by FcγR expression assessment using flow cytometry. To stabilize HIF-1α and HIF-2α in primary mononuclear phagocytes and THP-1 cells, HIF prolyl hydroxylase (HIF-PH) inhibitors were utilized as hypoxia mimetics.DMOG was purchased from EMD Millipore, directly dissolved in sterile PBS to a stock concentration of 20 mg/ mL, then filter sterilized and stored aliquoted at -80 °C.Roxadustat (FG-4592) was purchased from Stratech Scientific Ltd, dissolved in DMSO to create 50 mg/mL stock solution aliquots; these were further diluted in sterile PBS to 1 mg/mL, and stored aliquoted at -80 °C.To determine whether the stabilization of hypoxia inducible factors leads to FcγRIIb upregulation on monocytes, human PBMCs, purified monocytes or MDMs were incubated with 20 µM DMOG or 20 µM Roxadustat in complete CTL medium, at 37 °C and 5% CO 2 for 24 or 48 h, before flow cytometric analysis.THP-1 cells were treated with 0-200 µM DMOG. To assess the role of HIFs and Activator protein 1 (AP-1) in the upregulation of FcγRIIb in human monocytes and MDMs the following reagents were utilized to impair function or reduce expression of HIFs and or AP-1 transcription factor complex protein; c-Jun.In Additional file 5: Fig. S5a-b HD PBMCs were treated with 10 µM Digoxin (Sigma-Aldrich).In Fig. 5i LD PBMCs were treated with VHL inhibitor; VH298 [38], at 25 µM (Sigma-Aldrich).In Fig. 5j LD PBMCs were simultaneously treated with 20 µM DMOG and 20 µM of the HIF-α inhibitor; FM19G11 [39], (Sigma-Aldrich).To impair JNK/c-Jun interactions in Fig. 5k, LD PBMCs were treated with 20 µM DMOG and were simultaneously treated with 1 mM c-Jun peptide (R&D Systems [40]).All inhibitor treated cells were cultured in complete CTL medium for 24 h, followed by FcγR expression assessment using flow cytometry.M1 MDMs were Fig. 1 FcγR expression profiling of human PBMCs cultured at low or high density for 48 h.a, Expression of FcγR on primary human monocytes (FSC hi CD14 + cells) in low density (LD) or high density (HD) PBMC cultures determined using flow cytometry.Representative histograms above and quantified for 11 independent healthy donors below.b, Comparison of FcγR activating:inhibitory (A:I) ratio between LD and HD monocytes, (n = 11 per group).c, Quantification of FcγR and myeloid cell surface markers on monocytes in LD and HD PBMC cultures determined using flow cytometry and PE fluorescence quantitation beads, group means ± SD are shown, (n = 5 per group).d, Assessment of FcγRIIb expression by Western blot in LD and HD monocytes using CHO cells transfected with FcγRIIb1, and FcγRIIb2 isoforms as controls.e, Combined Western blot data of FcγRIIb expression normalized to HSC70 loading control (left) and fold change of FcγRIIb expression relative to HSC70 in LD and HD monocytes (right), (n = 16 per group).Each data point represents a unique healthy adult donor.Statistical significance between groups was assessed using a paired two-tailed Wilcoxon test (*p < 0.001, *p < 0.0001 and ns = non-significant).Also see Additional file 1: Fig. S1 (See figure on next page.)treated with c-Jun peptide with or without simultaneous treatment with 20 µM DMOG for 48 h.These M1 MDMs were cultured in complete RPMI 1640 with M1 skewing agents (IFN-γ and LPS) and incubated at 37 °C and 5% CO 2 , followed by FcγR expression assessment using flow cytometry.siRNA For siRNA manipulation of monocytes, immediately after isolation from PBMC samples, monocytes were washed in PBS, centrifuged at 300 × g for 5 min, supernatant discarded and the cell pellet resuspended at 3 × 10 7 cells/mL in buffer T (ThermoFisher).A NeonTM tube was filled with 3 mL of buffer E2 and inserted into the NeonTM Pipette Station.HIF1Α, HIF2A, JUN or Silencer ™ negative control siRNA (ThermoFisher) were added to the isolated monocyte/buffer T mixture so that the final working concentration of siRNA was 100 nM.The siRNA/monocyte/ buffer T mixture was taken up into a 100 µL NeonTM Pipette tip (ThermoFisher) and electroporated with the Neon transfection system (ThermoFisher) using the settings: 1920 V, 25 ms, 1 pulse.The electroporated cells were either shared across 2 wells of a 24-well plate with 1 ml of antibiotic free media (CTL medium + 10% FCS) for LD culture; or placed in a single well of a 96-well plate with 100 µl antibiotic-free media for HD culture.Electroporated cells were incubated in antibiotic-free CTL medium for 48 h before FcγR expression was analyzed by flow cytometry. ", "section_name": "Cell culture", "section_num": null }, { "section_content": "Human immune cells were first incubated in complete RPMI 1640 supplemented with 10% Human AB serum (Invitrogen) and incubated at room temperature for 15 min and then centrifuged at 300 × g for 5 min.Cells were then resuspended at 10 × 10 6 cells/mL in flow cytometry wash buffer (PBS with 1% w/v BSA (Europa), 0.1% w/v sodium azide (Sigma-Aldrich)). 1 × 10 6 PBMCs, purified monocytes, MDMs, THP-1 cells, immune cells isolated from pleural fluid, lymphocele, RCC or healthy (normal) kidney tissue in 100 µL were stained with fluorophore-conjugated mAbs per FACS tube, for 30 min at 4 °C.Samples were stained with anti-CD3 PerCP (clone: SK7), anti-CD56 PE (clone: HCD56), anti-CD19 APC-Cy7 (clone: HIB19), anti-CD14 Pacific Blue (clone: M5E2), anti-CD163 PE-Cy7 (clone: RM3/1), anti-HLA-DR APC-Cy7 (clone: L243), anti-CD40 APC-Cy7 (clone: 5C3), anti-human CD11b PE (clone: ICRF44), anti-human CD274 (B7-H1, PD-L1) PE (clone: MIH3) or IgG1κ-FITC (clone: MOPC-21) isotype control (all from BioLegend).FcγR staining was carried out using anti-FcγRI FITC (clone: 10.1, F(ab') 2 ), anti-FcγRII PE (clones: AT10 and KB61), anti-FcγRIIa FITC (clone: E08, F(ab') 2 ), anti-FcγRIIb FITC (clone: 6G11, F(ab') 2 ), anti-FcγRIIb FITC (clone: 2B6, F(ab') 2 ), anti-FcγRIIIa FITC (clone: 3G8, F(ab') 2 ), and isotype control human IgG1 FITC (clone: FITC8 F(ab') 2 ) were all generated from published sequences in-house or provided by BioInvent International AB.The FcγR activating:inhibitory (A:I) ratio was calculated by summing up the Geometric mean (Geomean) fluorescent intensities of the activating FcγR (FcγRI, FcγRIIa and FcγRIIIa) staining and dividing by the Geomean fluorescent intensity of FcγRIIb staining.Alternatively, anti-FcγR mAbs were used in an APC format.In order to determine expression levels of the hypoxia marker; Carbonic Anhydrase IX, anti-CAIX (clone: M75), mouse IgG1, Fc Silent ™ , Kappa APC (Absolute antibody) was used.Cells were then washed with flow cytometry wash buffer and if pre-cultured with translation, transcription, HIF-PHD, HIF-α or c-Jun inhibitors, cells were also stained with propidium iodide (PI, Sigma-Aldrich) before analysis of the cells by flow cytometry, in order to determine cell viability.HIF-1α and GLUT1 were measured using the Hif1α + GLUT1 Hypoxic Response Human Flow Cytometry Kit (Abcam). 1 × 10 6 cells LD or HD monocytes were harvested and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 15 min and pelleted.The pellet was resuspended in 90% ice cold methanol and incubated at -20 °C for at least 30 min.Within 1 week of fixing, cells were warmed to room temperature and the methanol removed before rehydrating and washing cells in blocking buffer.After incubating cells for 30 min in blocking buffer the primary mouse HIF-1α and rabbit GLUT1 antibodies were added (Table 1).After 1 h, the cells were washed in PBS and the appropriate secondary antibodies anti-mouse Alexa Fluor 488 (A488), (in house) and F(ab') 2 Donkey anti-Rabbit IgG PE (Affymetrix eBioscience), were added to the cells and allowed to incubate for 1 h.Cells were washed in flow cytometry wash buffer before analysis as stated above.Results of FcγR expression are shown as geometric mean fluorescent intensity (MFI) for FcγR expression on single/live B cells (FSC -A lo SSC -A lo CD19 + CD3 -), NK cells (FSC -A lo SSC -A lo CD3 -CD56 dim ), monocytes (FSC -A hi SSC -A lo CD14 lo/intermediate/high ) and tumor-associated macrophages (FSC-A hi SSC -A lo CD3 -CD56 -CD19 -CD14 -CD163 + ) (see Additional file 1: Fig. S1a-b for flow cytometric gating strategy for all human immune cells).FcγR, CAIX, CD40, PD-L1 and HLA-DR expression levels were corrected by subtracting the geometric MFI of the corresponding isotype control.Post-staining with fluorophore-conjugated mAbs, cells were analyzed using BD FACSCantoII or FACSCalibur (BD Biosciences) flow cytometers and data analyzed using FlowJo Version 10 software (FlowJo LLC). ", "section_name": "Flow cytometry of human cells", "section_num": null }, { "section_content": "Murine immune cells (from peripheral blood, peritoneum, spleen or bone marrow) were resuspended at 1 × 10 7 cells per mL in flow cytometry wash buffer.S7a for flow cytometric gating strategy for murine immune cells.FcγR expression levels were corrected by subtracting the geometric MFI of the corresponding isotype control.Post-staining with fluorophore-conjugated mAbs, cells were analyzed using BD FACSCantoII or FACSCalibur (BD Biosciences) flow cytometers and data analyzed using FlowJo Version 10 software (FlowJo LLC). ", "section_name": "Flow cytometry of murine cells", "section_num": null }, { "section_content": "Isolated monocytes (5 × 10 6 ) were collected and centrifuged at 800 × g for 5 min at room temperature.The supernatant was removed, and the resulting cell pellet was lysed using 20 µL of RIPA buffer (Abcam) supplemented with a Western blot protease inhibitor cocktail (Abcam).The lysed cells were stored at -20 °C.The protein concentration of the lysed cells was determined using a Bradford assay; 50 µg of protein was added to 5 µL of laemmli buffer (Abcam), and distilled H 2 O was added to make each sample a total of 20 µL.The protein-laemmli buffer mix was then heated at 95 °C for 5 min.The samples were loaded onto pre-made 10%, 1.50 mm × 10 well, bis-tris gels (NuPage R) and run at 150 V. Proteins were transferred onto nitrocellulose blotting membranes (GE Healthcare life sciences) in a transfer cassette run at 30 V for 90 min.The proteins probed for were FcγRIIb and HIF-1α and/or HIF-2α and/or JUN with HSC70 as a loading control.The membrane was blocked in a 5% BSA Tris-buffered saline-tween (TBS-T), 0.01% azide solution for one hour.Anti-FcγRIIb (clone:EP888Y, Abcam), anti-HIF-1α (clone: polyclonal, Novus Biologicals) and anti-HSC70 (clone: B-6, Santa Cruz Biotechnology) were added at a 1:500 dilution, anti-HIF2-α (clone: D6T8V), anti-c-Jun (clone: 60A8), anti-phospho-c-Jun (clone: D47G9) and anti-phospho-c-Fos (clone: D82C12, all from Cell Signalling Technology) were added at a 1:1000 dilution.The antibodies were left on the nitrocellulose membrane overnight at 4 °C.The next day, the blots were washed in a 5% BSA Tris-buffered saline-tween (TBS-T) solution followed by a 1 h incubation with horseradish peroxidase (HRP)-linked secondary antibodies.An ECL Western Blotting substrate (Pierce R) was used to detect HRP activity and imaged using the Imager Chemi Doc-It Imaging system (UVP) and the VisionWorks RLS software (UVP).The images were quantified using ImageJ 1.4.3.67 software. ", "section_name": "Western blotting", "section_num": null }, { "section_content": "PBMCs or purified monocytes were cultured in serum free complete CTL medium in 24-well plates with integrated oxygen sensors (OxoDish-R-DW, PreSens) and placed on the OxoDish R sensor dish reader (SDR) (Pre-Sens) to measure oxygen levels in the culture conditions over a 24 h period, as previously described [41,42].The reader and plates were placed at a constant humidity, 37 °C, 5% CO 2 and oxygen levels were measured every 10 min for 24 h. ", "section_name": "Oxygen sensing (SensorDish Reader)", "section_num": null }, { "section_content": "LD and HD human PBMCs or isolated monocytes were cultured for up to 48 h in 24-well plate in serum free CTL medium.Supernatants from these cultures were analyzed for CO 2 , metabolites, pH and Oximetry using an ABL 835 FLEX blood gas analyzer (Radiometer Medical ApS). ", "section_name": "Radiometer analysis of cell culture media", "section_num": null }, { "section_content": "ChIP was performed on magnetically sorted isolated monocytes (Pan Monocyte Isolation Kit, human, Miltenyi Biotech), cultured for 10 h at LD with or without 20 µM DMOG, as previously published with several modifications (Hayakawa et al., 2004).Briefly, the Sim-pleChIP Enzymatic Chromatin IP Kit (Magnetic Beads), (Cell Signalling Technology), was used; 4 × 10 6 untreated or DMOG treated monocytes were fixed using 37% formaldehyde for 15 min at room temperature.Monocytes were then washed twice in ice cold PBS, centrifuged at 2000 × g for 4 min at 4 °C, supernatant discarded and the dried pellets stored at -80 °C overnight.Cell pellets were then thawed on ice and treated with sodium dodecyl sulfate (SDS) containing buffers as per the manufacturer's protocol.DNA in each sample was digested using 0.5 µL Micrococcal Nuclease per immunoprecipitation (IP) and incubated for 15 min at 37 °C.The digestion was stopped using 0.5 M ethylenediaminetetraacetic acid (EDTA) and after further washing and treatment with buffers as per the manufacturer's protocol, the digests sonicated using a Soniprep 150 sonicator (MSE), at setting 3 for 15 cycles of 45 s on and 15 s off, whilst being kept on ice.The lysates were then clarified by centrifugation at 9,400 × g for 10 min at 4 °C.The supernatants were removed and stored at -80 °C overnight.50 µL of this sample was run on a 1% agarose gel following digestion with RNAse A and Proteinase K, as per the manufacturers protocol using the SimpleChIP Enzymatic Chromatin IP Kit, (Cell Signalling Technology).DNA for all samples was observed to be fragmented between 150-900 bp.Each DNA sample was then incubated overnight at 4 °C with Rabbit mAb IgG XP isotype control (clone DA1E) for the negative control IP, Histone H3 XP Rabbit mAb (clone: D2B12) for the positive control, c-Jun Rabbit mAb (clone: 60A8), HIF-1α XP Rabbit mAb (clone D1S7W) or HIF-2α Rabbit mAb (clone: D6T8V, all mAbs purchased from Cell Signalling Technology). The chromatin from each sample was then separated from the aforementioned mAbs using protein G magnetic beads followed by reversal of chromatin/DNA cross linking and DNA purification as per the manufacturer's protocol.Using real-time quantitative polymerase chain reaction (RT-QPCR) RPL30; the positive control gene was amplified from DNA isolated via the anti-Histone H3 ChIP assay.Commercially available primers (Cell Signalling Technology) and the SimpleChIP Universal qPCR Master Mix (Cell signalling Technology) were then used to detect RPL30 as per the manufacturer's protocol.Using the same SimpleChIP Universal qPCR Master Mix the FCGR2B promotor region was also amplified at specific regions predicted to contain AP-1 or HIF-α binding motifs within the 1 Kb gene promotor upstream of the transcription start site (TSS), using custom-designed primers (see Additional file 1: Table S1 for list of primers, purchased from Integrated DNA Technologies). ", "section_name": "Chromatin immunoprecipitation (ChIP) Assay", "section_num": null }, { "section_content": "Monocytes were isolated from PBMC cultures (using the Pan Monocyte Isolation Kit, human, Miltenyi Biotech), cultured at HD, and harvested at 0, 2, 10 and 24 h from 2 donors.B cells were also isolated from PBMC cultures (using the B cell Isolation Kit II, human, Miltenyi Biotech), cultured at HD and harvested at 0 and 24 h.Total RNA was isolated from these cells using the RNAeasy Mini Kit (Qiagen) for assessment on the whole genome DASL array. A whole genome DASL array was carried out on monocyte and B cell total RNA samples.The resulting dataset was corrected for background using negative controls and normalized using the neqc function of the limma v3.24.15 Bioconductor package [43] in R v3.3.2 (R Core Team (2017).R: A language and environment for statistical computing.R Foundation for Statistical Computing, Vienna, Austria.URL https:// www.R-proje ct.org/).The dataset was also quality checked by only including probes that were expressed in at least 3 arrays according to detection p-values of 5%.Multi-dimensional scaling plots were generated using the plotMDS function to look at the variability between donors and time points.The normalized, quality checked data was then used to evaluate differentially expressed genes between the 4 time points and a cut-off based on a defined FDR was used to generate a list of gene candidates between every iteration of comparisons.These identified genes were used to generate heatmaps in Ingenuity Pathway Analysis (IPA) v01-07.Gene set enrichment analysis was performed using the fgsea R package (Korotkevich, 2019, https:// doi.org/ 10. 1101/ 060012) and referenced to Broad hallmark gene sets (h.all.v7.2).Genes were pre-ranked using a signed log 10 -transformed FDR from differential analysis in DESeq2 (Love, 2014), with the sign denoting the direction of logFC. ", "section_name": "Whole genome DASL (cDNA-mediated annealing, selection, extension and ligation) Array and bioinformatic analyses", "section_num": null }, { "section_content": "Monocytes were isolated from PBMCs using the Pan Monocyte Isolation Kit, human, (Miltenyi Biotech), from 7 different healthy adult donors and cultured at LD (1 × 10 6 /mL in a 24-well plate) in the presence or absence of 20 μM DMOG, in complete CTL medium at 37 °C and 5% CO 2 .Complete CTL medium was removed from monocyte cultures and they were disrupted using QIAzol lysis reagent (Qiagen) and total RNA was then isolated using the miRNeasy mini kit (Qiagen) as per the manufacturer's protocol at 0, 2, 10 and 24 h post-culture.RNA quantity and quality for each sample was assessed using the RNA 6000 Nano kit (Agilent), analyzed using a Bioanalyzer (Agilent) and only samples with RIN scores > 8 were used for further downstream analysis.RNA samples were enriched for mature, poly-A mRNA transcripts and 150 base pair paired-end sequencing was carried out on the NovaSeq 6000 (Illumina) platform by Oxford Genomics Centre (Oxford, UK) resulting in an average of 39 million reads per sample.Sequencing reads were aligned to the human genome (primary assembly, GRCh38.p12,Ensembl) [44,45] using the STAR alignment algorithm [46] and uniquely-mapped alignments overlapping gene exons were counted using feature-Counts from the Rsubread package [47].Counting was performed relative to Ensembl 97 (Jul 2019) gene annotation and counts summarized at the gene level.Gene expression filtering and normalization was carried out in R, using edgeR [48].Genes with below-threshold counts were filtered out (filterByExpr: min.count= 30, min.count.total= 45) and between-sample normalization was performed using the trimmed mean of M-values method.Gene expression is reported as counts-per-million (CPM). ", "section_name": "RNA-Seq", "section_num": null }, { "section_content": "Differential expression analysis was carried out using the limma [43] linear-modelling R package and specifically the voomWithQualityWeights function to provide geneand sample-specific weights to account for mean-variance relationships in the data.A group-means approach was taken for the design matrix, with donor as a blocking variable.Between-treatment comparisons were made at 2, 10, and 24 h for DMOG versus untreated and acrosstime comparisons were also made for the following between-treatment comparisons; 10 h versus 2 h and 24 h versus 10 h.Differences in expression across-time were also assessed within-treatment for DMOG and untreated separately; 2 h versus 0 h, 10 h versus 2 h and 24 h versus 10 h.Differential expression tests were performed for each comparison with a null interval hypothesis for the expression fold change (FC) [-log2(1.2) < log2(FC) < log2-(1.2)]with false discovery rate (FDR) < 0.05 per comparison using the Benjamini-Hochberg procedure.Principal Component Analysis was performed using genes that are differentially expressed (in either direction) in at least one of the between-treatment comparisons. ", "section_name": "RNA-Seq differential gene expression", "section_num": null }, { "section_content": "ATAC-seq was performed as previously described [49][50][51], with minor alterations.Monocytes sourced from healthy adult donors were isolated from PBMCs using the Pan Monocyte Isolation Kit, human, (Miltenyi Biotech) and cultured in complete CTL medium at 37 °C in 5% CO 2 .For the experiment comparing LD and HD culture conditions, monocytes from 3 donors were either plated at 1 × 10 6 cells/mL (LD) or 1 × 10 7 cells/mL (HD) and cultured for 24 h.For the time course experiment assessing DMOG treatment, monocytes from 7 donors were cultured at 1 × 10 6 cells/mL either with or without 20 µM DMOG for 24 h.ATAC-seq library preparation was the same in both cases: 50,000 monocytes per sample were harvested at 24 h post-culture and centrifuged at 300 × g for 5 min at 4 °C.The cell pellet was carefully resuspended in transposase reaction mix (12.5 μL 2 × TD buffer, 2 μL TDE1 (Illumina)), 10.25 μL nuclease-free water and 0.25 μl 1% digitonin (Sigma-Aldrich) per sample, for 30 min at 37 °C.11 μL of DNA was isolated from each sample using the MiniElute PCR Purification Kit (Qiagen).1 μL of eluted DNA from each sample was used in a quantitative PCR (qPCR) reaction to estimate the optimum number of amplification cycles.The remaining 10 μL of each library was amplified for the number of cycles corresponding to the C q value from the qPCR (the cycle number at which fluorescence has increased above background levels).Library amplification was followed by Solid Phase Reversible Immobilization (SPRI, Beckman Coulter) size selection to exclude fragments > 1,200 bp.DNA concentration was measured with a Qubit fluorometer (Life Technologies) and library amplification was performed using custom Nextera primers [49].Libraries were sequenced by the Biomedical Sequencing Facility at CeMM (Vienna, Austria) using the Illumina HiSeq 3000/4000 platform.50 bp single-end sequencing was performed for the LD-HD comparison experiment with two technical replicate sequencing runs per sample library.75 bp paired-end sequencing was performed for the DMOG time course experiment. ", "section_name": "ATAC-seq (assay for transposase-accessible chromatin using sequencing)", "section_num": null }, { "section_content": "For the LD-HD comparison experiment, sequencing reads were aligned to the human genome (GRCh38) using HISAT2 v2.1.0[52] and the non-default parameter -no-spliced-alignment.Peaks were called for each sample and technical replicate separately using MACS2 v2.2.1 [53], callpeak function with non-default parameters -nomodel -shift -100 -extsize 200 -B -broad.For the DMOG time course experiment, sequencing reads were trimmed of adapter sequences using cutadapt v2.4 (Marcel Martin, 2020, https:// doi.org/ 10. 14806/ ej. 17. 1. 200) and aligned to the human genome (GRCh38) using HISAT2 v2.1.0and the non-default parameter -nospliced-alignment. Peaks were called for each sample separately using MACS2 v2.2.6 callpeak function with non-default parameters -nomodel -call-summits.For both experiments, read counting in peaks was performed in R with the diffbind package for differential open region calling [54], using only uniquely-mapped reads and the DEseq2 option [55].Peak annotation was performed using the ChIPseeker package in R [56] and GENCODE 32 annotation [57]. For the DMOG time course experiment transcription factor (TF) binding sites that overlapped peaks were determined using the Open Regulatory Annotation database (ORegAnno) 3.0 [58] and the bedtools function closest [59].Differentially open peaks between DMOG-treated or untreated monocytes were scanned for TF binding sites and the occurrence frequency of each TF over all differentially open peaks was calculated.To determine if these frequencies were significant or obtained randomly, the same number of regions as differentially open peaks were selected randomly from the genome and scanned for the same TFs.This process was repeated 1000 times to a generate a per-TF frequency distribution for randomly selected regions.Z-scores were calculated for the observed TF frequency in differentially open regions with respect to the random region frequency distribution. ", "section_name": "ATAC-Seq data analysis", "section_num": null }, { "section_content": "Publicly available FASTQ files for samples from Tausendschon et al. [60], (GSE43109), were aligned to the human genome hg19 using bowtie (v1.1.2,pre-built hg19 bowtie index: https:// benla ngmead.github.io/ aws-index es/ bowtie) [61], with the following alignment reporting parameters; -k 4 -m 4 -best.This allows for multi-mapping alignments which occur in the FCGR low affinity locus due to sequence homology.The multi-mapping alignment positions were checked for those reads that aligned to peaks approximately 10 Kb upstream of FCGR2B and FCGR2C transcriptional start sites.These reads only multi-map between homologous sequences of FCGR2B and FCGR2C and not elsewhere on the genome. ", "section_name": "Chromatin immunoprecipitation-Seq data analysis", "section_num": null }, { "section_content": "Whole blood samples were sourced from Rhesus D positive healthy adult female donors.Monocytes were also isolated from these samples using density centrifugation (Lymphoprep) and the Pan Monocyte Isolation Kit, human (Miltenyi Biotech).Monocytes were then cultured in complete RPMI for 48 h at LD and HD as previously described.Whole blood samples were taken again from the same Rhesus D positive donors and RBCs isolated using density centrifugation (Lymphoprep).RBCs were then were labelled with 2 µM Carboxyfluorescein succinimidyl ester (CFSE), quenched with complete RPMI and washed twice in complete RPMI.These RBCs were then opsonized with 5 ug/mL anti-Rhesus D hIgG1 antibody or cetuximab isotype control in flow cytometry wash buffer and washed once.5 × 10 5 CFSE labelled and antibody pre-treated RBCs were cultured with 1 × 10 5 autologous LD or HD monocytes (5:1 target:effector ratio) in 100 uL complete RPMI per well, in a 96-well plate.Alternatively, mAb-opsonized and CFSE-labelled RBCs were cultured with autologous LD or HD monocytes pre-treated with E08 (anti-human-FcγRIIa) or 6G11 (anti-FcγRIIb) F(ab') 2 fragments to block IgG Fc-FcγRII interactions.After a 2 h incubation period at 37 °C, and 5% CO 2 , the monocytes were stained with anti-CD14 Pacific Blue (Biolegend), washed once, and CD14 + CFSE + (Monocytes that had phagocytosed RBCs) of total CD14 + cells were quantified using a BD FACSCantoII (BD Biosciences) flow cytometer and data analyzed using FlowJo Version 10 software (FlowJo LLC). ", "section_name": "RBC phagocytosis assay", "section_num": null }, { "section_content": "MDM phagocytic function was assessed as reported previously [62].In brief, M0, M1 or M2 macrophages were seeded at 1 × 10 5 cells per well in a flat bottomed 96-well plate in 100 µL of complete RPMI.CLL cells were used as targets and labelled with 5 µM CFSE for 10 min and washed with complete RPMI.CLL cells were opsonized with Herceptin or cetuximab (negative control) and rituximab or obinutuzumab, incubated at 37 °C and 5% CO 2 for 30 min.Opsonized target cells were then washed and added to the MDMs at an effector to target ratio of 1:5, incubated at 37 °C and 5% CO 2 for 2 h.Cells in each well were labelled with anti-human FcγRIIIA-APC (BioLegend), with target uptake determined using the BD FACSCantoII or FACSCalibur (BD Biosciences) flow cytometers and analyzed using FlowJo Version 10 software (FlowJo LLC). ", "section_name": "MDM antibody-dependent cellular phagocytosis (ADCP) assay", "section_num": null }, { "section_content": "WT C57BL/6 J or BALB/c mice were inoculated with MCA205, EG7 or CT26 tumor cells (5 × 10 5 ) subcutaneously (s.c.) into the right flank and mice sacrificed once tumors sizes reached 500 mm 3 .Tumors and spleens were then harvested and FcγR expression on monocytes and macrophages in these tissues determined using flow cytometry. Additionally, WT C57BL/6 J mice were inoculated with E0771 tumor cells (2.5 × 10 5 , injected into mammary fat pad) and mice sacrificed once tumors sizes reached 500 mm 3 .To determine hypoxic regions within these tumors, Hypoxyprobe (pimonidazole), Hypoxyprobe-RedAPC Kit (Hydroxyprobe ™ ) was resuspended at a concentration of 30 mg/mL in 0.9% sterile saline.Multiple mice (n = 5 per tumor type) were injected intravenously (i.v., tail vein) with 60 mg/kg of the pimonidazole solution.Mice were sacrificed 90 min later, tumors harvested, embedded in OCT (CellPath, Newtown, Powys, U.K.) and frozen in isopentane on a bed of dry ice.Tumor samples were stored in plastic scintillation vials at -80 °C [63].In order to assess the effects of HIF-PH inhibitor treatment on FcγR expression on myeloid cells in vivo, gender-and age-matched WT C57BL/6 J mice were dosed with 4 mg DMOG or PBS vehicle control via intraperitoneal (i.p.) route on three consecutive days.Mice were sacrificed 24 h later, the blood and peritoneal lavage harvested and FcγR expression levels assessed on monocytes, neutrophils and macrophages, using flow cytometry.WT C57BL/6 J mice were also dosed with 200 µg Roxadustat or PBS control i.p. on three consecutive days.Mice were sacrificed 24 h later, the blood, peritoneal lavage, spleen and bone marrow were harvested and FcγR expression levels assessed on neutrophils, monocytes and macrophages in each compartment, using flow cytometry. In order to assess whether HIF-PH inhibitors can impair mAb-mediated target cell depletion in vivo, age-and gender-matched WT C57BL/6 J mice were treated with 200 µg Roxadustat or PBS i.p. on two consecutive days.20 h later 10 µg of anti-mouse CD20 mAb; 18B12 or isotype control mAb (DB7/12) was given i.v.Mice were treated again with 200 µg of Roxadustat or PBS i.p. before peripheral blood was taken (tail bleed) to assess systemic levels of CD19 + cells 24 h later, using flow cytometry.In separate experiments, age-and gender-matched WT C57BL/6 J mice were dosed with 200 µg Roxadustat or PBS i.p. on two consecutive days prior to receiving CFSE labelled EL4-huCD20 + cells i.p. on the second day.On the third day mice were treated with 50 ug of rituximab or cetuximab i.v.followed by a final treatment with 200 µg of Roxadustat or PBS also on the third day.EL4-hCD20 + cell depletion in the peritoneal lavage following mAb treatment was quantified using flow cytometry 24 h later. In order to assess the effects of HIF-PH inhibitor treatment on human FcγRIIb expression on myeloid cells and systemic B cell depletion in vivo, Tg hFcγRIIb +/-x mFcγRII -/-x hCD20 +/-mice were dosed with 4 mg of DMOG or PBS vehicle control i.p. on two consecutive days.Mice were treated with 50 µg rituximab or cetuximab i.v.followed by a final treatment with 4 mg DMOG or PBS also on the third day.Mice were sacrificed 24 h later with peritoneal lavage, spleen, and bone marrow harvested.FcγR expression levels were assessed on monocytes, neutrophils, and macrophages and frequencies of live B (CD19 + ) cells quantified using flow cytometry. In order to assess the effects of HIF-PH inhibitor treatment on specific hCD20 + B cell depletion in vivo, an adoptive transfer assay was performed as before [30]: Tg hFcγRIIb +/-x mFcγRII -/-mice were dosed with 4 mg of DMOG or PBS vehicle control i.p. on three consecutive days.On the third day mice were given 3 × 10 6 target splenocytes from hCD20 +/-x mFcγRII -/-mice and 3 × 10 6 non-target splenocytes from WT C57BL/6 J mice differentially labelled with CFSE, i.v.These mice were again treated with 4 mg of cetuximab or PBS i.p. prior to receiving 50 µg rituximab or cetuximab 24 h later.Depletion of target and non-target splenocytes in the spleen was quantified using flow cytometry.Finally, Tg hFcγRIIb +/-x mFcγRII -/-mice were treated with 4 mg DMOG or PBS i.p. on three consecutive days, prior to receiving CFSE labelled EL4-huCD20 + cells i.p. on the third day.These mice were then treated with 50 µg Rituximab or Cetuximab i.v.followed by a final treatment with 4 mg DMOG or PBS on the fourth day.EL4-huCD20 + cell depletion in the peritoneal lavage following mAb treatment was quantified using flow cytometry 24 h later. ", "section_name": "In vivo studies", "section_num": null }, { "section_content": "Fresh murine spleen and CT26, MC38 or E0771 tumor tissue were embedded in OCT (CellPath, Newtown, Powys, U.K.) and frozen in isopentane on a bed of dry ice.Sections (8 μm) were cut, air-dried (overnight), fixed in 100% acetone for 10 min and blocked with 2.5% normal goat serum before incubation with anti-mouse FcγRII (clone: AT130-2, in house).Murine FcγRII was detected (45 min) with Alexa Fluor 488-conjugated anti-rat IgG (Life Technologies)).When using a second rat primary antibody, sections were incubated with rat IgG (50 μg/mL, 30 min, prepared in house).Hypoxic regions within tumor sections were detected by staining for pimonidazole using the Hypoxyprobe-RedAPC Kit (1000 mg pimonidazole HCl plus 2 units of 4.3.11.3 mouse Dylight ™ APC-Mab, Hydroxyprobe ™ ) as per the manufacturer's protocol.Sections were mounted in Vectashield Hardset (Vector Laboratories).Images were collected using a CKX41 inverted microscope with reflected fluorescence system equipped with a CC12 color camera running under Cell B software, using Plan Achromat 10 × 0.25 and 40 × 0.65 objective lenses (all from Olympus, Southend-on-Sea, Essex, U.K.).RGB image files (.tif ) were transferred to Adobe Photoshop (CS6; Adobe Systems, San Jose, CA) and all images treated in the same way.Tissue autofluorescence was removed by difference blending between the color channels and red/green image overlays contrast-stretched to use the whole grayscale.Colocalization analysis was performed using the Coloc2 plugin (https:// imagej.net/ plugi ns/ coloc-2) in Fiji [64].Background was measured in unstained areas of the section and the mean plus 2 times standard deviation subtracted from the image.Coloc2 was set to measure Manders coefficients using Costes threshold regression and a PSF of 3 and to perform a Costes significance test with 10 randomisations (Costes P value > 0.95 denotes non-random colocalization). ", "section_name": "Immunofluorescence microscopy", "section_num": null }, { "section_content": "Statistical significance when comparing FcγR expression in vitro on human immune cells on untreated versus treated cells or between groups of untreated and HIF-PHD inhibitor treated mice was determined between the groups using either an unpaired two-tailed t-test or paired two-tailed Wilcoxon tests.One-way analysis of variance (ANOVA) was used with the Bonferroni correction for multiple comparisons as needed, to compare multiple treatment groups when assessing mAb mediated cell depletion in vivo.As a large number of statistical tests have been carried out in a range of contexts, p-values should be interpreted with care and within the overall scientific context.Data analysis was carried out using the Graphpad Prism version 8.0.1 software.Statistical significance defined as p < 0.05, p < 0.01 * p < 0.001 and **** p < 0.0001 and ns = non-significant. ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Leukocyte-based in vitro assays remain one of the gold standards for determining mAb efficacy and predicting adverse responses in patients [65].We have previously characterized several assay formats reporting on the impact of FcγRs on mAb mediated immune cell responses in vitro [32,66].This work led us to observe that when human primary monocytes are cultured at high density (HD; ~ 8 × 10 6 cells per cm 2 ) they markedly upregulate the inhibitory Fc receptor for IgG; FcγRIIb [32].FcγRIIb impairs myeloid cell effector functions by antagonizing activating FcγRs and so we sought to further understand how high cell density modulates FcγR gene and protein expression.Concordant with our previous findings [32] the high affinity activating IgG receptor, FcγRI, remained unaltered under HD conditions, whereas the expression of the low affinity FcγRs; FcγRIIa, FcγRIIb and FcγRIIIa were all significantly increased on monocytes in HD PBMC cultures relative to monocytes from LD cultures (LD; ~ 0.8 × 10 6 cells per cm 2 ), 48 h postculture (p < 0.0001 for all low affinity FcγR comparing LD versus HD monocytes).Although, there were significant increases in FcγRIIa (~ twofold) and FcγRIIIa (sevenfold), HD culture induced a striking ~ 110-fold increase in FcγRIIb expression on monocytes (Fig. 1a).Relative quantification of FcγR expression using PE-conjugated beads, allowed us to ascertain that HD monocytes had a tenfold lower activating to inhibitory (A:I) FcγR expression ratio in comparison to LD monocytes (Fig. 1b).The full extent of the profound FcγRIIb upregulation was highlighted when we compared its expression to other monocyte cell surface markers, including MHC class I/ II molecules, CD33, CD11b, CD14, CD83 and CD86, showing it to be significantly higher (p < 0.01, comparing FcγRIIb versus MHC class I expression levels, Fig. 1c).Western blotting confirmed that both FcγRIIb1 and FcγRIIb2 isoforms [67] were upregulated with FcγRIIb2 the dominant isoform expressed (Fig. 1d-e).Upregulation of FcγRIIb on HD monocytes was confirmed using multiple anti-FcγRIIb and pan FcγRII specific mAbs (Additional file 1: Fig. S1c).When comparing LD versus HD cultures, there was no significant difference in the expression of FcγRIIb on B cells, nor FcγRIIIa on NK cells, which was confirmed by flow cytometry PE bead quantification, demonstrating that FcγR expression on these cell subsets remained unaffected under HD culture (Additional file 1: Fig. S1d).CD3 + T cells remained FcγR negative regardless of cellular density (data not shown).In conclusion, we observed that although all low affinity FcγRs were upregulated on monocytes, this was not observed on other cell types within the same PBMC culture and the most striking feature was the marked and specific upregulation of the inhibitory FcγRIIb on the monocytic population. ", "section_name": "High density cell culture elicits marked induction of FcγRIIb expression on human monocytes", "section_num": null }, { "section_content": "Next, we performed a transcriptomic characterization of HD monocytes to investigate gene expression changes which were associated with the enhancement of monocyte FcγRIIb expression.A whole genome DASL array was carried out on donor matched samples of fresh purified peripheral blood monocytes (M0) versus monocytes cultured at HD for 2 h (M2), 10 h (M10) and 24 h (M24), alongside fresh purified human B cells (B0), and purified B cells cultured at HD for 24 h (B24), sourced from two healthy human donors.Multidimensional scaling plots of gene expression revealed considerable divergence between fresh monocytes versus 24 h cultured HD monocyte samples, with this observation being mirrored by fresh B cells versus 24 h cultured B cell samples, indicating marked global gene expression changes occurring under HD culture in both immune cell subsets (Additional file 2: Fig. S2a).Gene set enrichment analysis (GSEA) was performed to assess the biological processes associated with these differentially expressed genes.The top 20 statistically significant Hallmark pathway enrichment categories revealed a prominence of stress, inflammatory and, to a greater extent, hypoxia related processes in HD monocyte cultures.We observed a downregulation of the oxidative phosphorylation related gene expression signature and conversely an upregulation of hypoxia and glycolysis related gene expression in HD monocytes after 10 and 24 h (Fig. 2a).Owing to the prominence and importance of these changes in the TME, we chose to focus further on these aspects.GSEA confirmed that even within 2 h, gene expression in HD monocyte cultures was enriched for the Winter Hypoxia Metagene gene set (NES = 2.02 and FDR = 0, Fig. 2b)a gene set involved in the hypoxia pathway in the TME [68].Gene expression analysis also confirmed the upregulation of FcγRIIa, FcγRIIIa and particularly FcγRIIb on HD monocytes when compared to fresh monocytes (Fig. 2c).Ingenuity pathway analysis (IPA) of differential gene expression revealed that several genes associated with the hypoxia pathway, such as HIF1A, HIF2A (EPAS1), and ARNT (HIF1β) were amongst the top 50 upstream regulator genes and proteins in HD monocytes.In addition JUN, encoding c-Jun, a protein which forms the AP-1 transcription factor complex alongside c-Fos and which was previously reported to regulate FCGR2B gene expression [69] was also a prominent upstream gene regulator.Comparing the differentially expressed genes in HD monocytes with differentially expressed genes in hypoxic monocytes (monocytes cultured at 1% O 2 ) versus normoxic monocytes generated in a previous study [70], revealed high similarity in the upstream regulators of differentially expressed genes in HD monocytes and hypoxic monocytes (Fig. 2d).Interestingly, HIFs were significantly associated with upstream gene regulation in HD monocytes when compared to B cells at 24 h postculture.Furthermore, AP-1 or its constituent proteins did not feature amongst the top 50 most differentially expressed upstream regulators of gene expression in the monocyte versus B cell comparisons, indicating differential transcriptional responses to HD culture in the two immune cell types (Additional file 2: Fig. S2b).To directly confirm whether HD cultures of PBMCs and monocytes were indeed hypoxic, we measured % O 2 levels using an SDR SensorDish ® Reader and observed that O 2 levels dropped from 21 to 1% within 90 min in HD cell cultures, whereas in LD cell cultures O 2 levels remained ≥ 12% even after 24 h (Fig. 2e).We also observed a significant reduction in pH and corresponding increases in lactate levels in HD PBMC culture supernatants (Fig. 2f ), as well as significant reductions in acid base excess and corresponding reductions in HCO 3 -(P), (Additional file 2: Fig. S2c-d).Although glucose levels significantly decreased in HD cultures at 48 h, glucose levels remained high (> 9 mmol/L, at pre-diabetic levels [71]) in the culture media (Additional file 2: Fig. S2e).Electrolyte levels were not significantly altered when comparing HD versus LD culture supernatants (Additional file 2: Fig. S2fi).To further validate the rapid emergence of hypoxia in HD monocyte cultures, we measured expression levels of HIF-1α, the master transcriptional regulator of cellular hypoxia [72], by Western blotting and confirmed enhanced HIF-1α protein accumulation in HD monocytes (Fig. 2g and Additional file 2: Fig. S2j-k).Using flow cytometry, moderate but significant increases in HIF-1α expression levels were confirmed in HD monocytes relative to LD monocytes (p < 0.05).Furthermore, two HIF-1α target genes; Glucose transporter 1 (GLUT1) and Carbonic anhydrase IX (CAIX) were also significantly upregulated on HD monocytes (p < 0.001 and p < 0.0001, respectively; Fig. 2h-i).The latter two genes are established indicators of hypoxia, and ATAC-Seq analysis of HD monocytes further revealed increased openness of not only the HIF1A gene itself but additionally HIF responsive genes ENO2, GLUT1, and CXCR4 (Fig. S2i).These observations led us to hypothesize that hypoxia upregulates FcγRIIb expression in mononuclear phagocytes.Therefore, we next applied an immunophenotyping and integrative multi-OMIC approach to investigate the mechanism by which HIF activation may enhance FcγRIIb expression on mononuclear phagocytes. ", "section_name": "High cell density elicits a hypoxia related gene signature and metabolic perturbation in human monocytes", "section_num": null }, { "section_content": "HIFs are constitutively expressed in all cells, however, in the presence of molecular O 2 they are rapidly degraded through the action of HIF-PH enzymes.In the absence (See figure on next page.)Fig. 2 Transcriptional and physiological profiling of HD human monocytes.The transcriptome of fresh and HD human monocytes cultured for 2, 10 or 24 h was investigated using microarray analysis.a, Pre-ranked GSEA; genes were ranked according to their differential expression between monocytes at 2, 10 or 24 h post-HD culture and fresh monocytes.Twenty Hallmark gene sets (v7.2) were significantly overrepresented (FDR < 0.05).Upregulated gene expression is signified in red and downregulation in blue.b, Enrichment plot of the Winter hypoxia gene set in monocytes post-HD culture.c, Heat map of FCGR gene expression.d, Microarray gene expression data was acquired using fresh monocytes (M0) and monocytes at 2 (M2),10 (M10) and 24 (M24) hours post-HD culture as well as monocytes cultured under hypoxic conditions (1% O 2 ) for 24 h (Bosco et al., 2006).Heat map of activation z-scores for the top 50 genes and proteins determined to be the most activating or inhibiting.e, % O 2 in LD and HD cultures of human PBMCs and isolated monocytes (n = 6 per group, thickness of lines for LD and HD represent SEMs for each time point).f, pH and Lactate in donor matched LD and HD PBMC culture supernatants (n = 5 per group).g, LD and HD monocyte cell lysates were generated and HIF-1α and HSC70 expression assessed using Western blotting.Representative Western blot staining for 2 donors is shown.h-i, Representative histograms and graphs showing expression of HIF-1α, CAIX and GLUT1 expression quantified using flow cytometry of LD and HD precultured monocytes (n = 11 per group).Each point on the graphs represents a unique healthy donor.Statistical significance between groups was assessed by using a paired two-tailed Wilcoxon test (p < 0.05, p < 0.001, p < 0.001 and *p < 0.0001).Also see Additional file 2: Fig. S2 of oxygen; i.e. hypoxia, HIF-PH-mediated hydroxylation of HIF-α subunits and their subsequent degradation is inhibited, allowing HIF-α to accumulate, facilitating its dimerization with HIF-1β (ARNT) and subsequent target gene binding and transcription [73].Endogenous HIF protein levels can be increased by the suppression of HIF-PH activity using small molecule competitive inhibitors of the HIF-PHs such as Dimethyloxalylglycine (DMOG) [74,75].Several studies have used DMOG as a hypoxiamimetic in mouse models of inflammation and LPS induced septic shock to alleviate pathology [76][77][78][79].We utilized DMOG for its previously reported ability to activate HIFs and induce downstream gene expression patterns that show concordance with those observed under physiological hypoxia [80].Peripheral blood monocytes from 7 adult healthy donors were sampled at 0 h (freshly isolated) or after 2, 10 or 24 h, post-culture with and without DMOG.Immunophenotyping using flow cytometry 24 h post-culture showed that DMOG treatment of LD monocytes resulted in a significant decrease in FcγRI expression (p < 0.001, Fig. 3a), significant increase in FcγRIIa expression (p < 0.01, Fig. 3b) and non-significant change in FcγRIIIa expression (Fig. 3d).However, as observed under HD conditions the most marked change was the pronounced enhancement of FcγRIIb expression in response to DMOG treatment (p < 0.001, Fig. 3c).Consequently, the FcγR A:I ratio was significantly decreased in DMOG treated monocytes (p < 0.05, Fig. 3e).RNA-Seq analysis was then carried out on these 7 healthy donor monocyte samples across the 24-h time-frame.Monocyte gene expression time course trajectories visualized by PCA using data from 6198 differentially expressed genes when comparing untreated versus DMOG treated monocytes, revealed considerable divergence at the 10 and 24 h time points between untreated versus treated samples, indicating large changes in the transcriptome of DMOG treated monocytes (Fig. 3f ).GSEA was performed to assess the biological processes associated with differentially expressed genes.The top 20 statistically significant Hallmark pathway enrichment categories revealed a prominence of stress, inflammatory and hypoxia related processes in DMOG treated monocytes (Fig. 3g).Importantly, we observed a downregulation of the oxidative phosphorylation gene set and conversely an upregulation of hypoxia, angiogenesis and glycolysis gene sets, in 10 and 24 h cultured DMOG treated monocytes (Fig. 3g).Formal correlative analysis of the HD and DMOG treated-monocyte cultures at 10 h postculture revealed a high degree of correlation amongst the enriched '50' Hallmark gene sets in both treatment types (Spearman's ρ = 0.73, p = 9.7e-09,Additional file 3: Fig. S3a).Further analysis of Log2 fold changes revealed that FCGR2B gene transcripts were differentially expressed at 10 h post-treatment with DMOG and remained elevated at 24 h post-treatment (Fig. 3h).Immunophenotyping of these monocyte samples using flow cytometry revealed cell surface expression changes in proteins concordant with the RNA-Seq transcriptional data; HLA-DR expression was significantly reduced on DMOG treated monocytes (p < 0.01 additional file 3: Fig. S3b).GSEA confirmed Winter Hypoxia Metagene gene set and Hypoxia gene set enrichment at 10 h post-treatment with DMOG (Fig. 3ij).We further assessed expression of genes which regulate glycolysis, angiogenesis and prolyl hydroxylases, showing these to be upregulated and coincident with increased expression of downstream targets; P4HA1, ENO1, GLUT1, VEGFA, EGLN1 and EGNL3.Furthermore, transcription factors ARNT and AP-1 encoding genes JUN and FOSL2 were also upregulated (Fig. 3k).HIF1A mRNA was itself downregulated (Fig. 3k), a known regulatory response to HIF-1α protein stabilization during hypoxia [81,82].The other major HIF; HIF2A (EPAS1) was not significantly differentially expressed in DMOG treated monocytes (Additional file 3: Fig. S3C).However, IPA was used to identify the top 50 upstream transcription factor regulators of differentially expressed Fig. 3 Transcriptional profiling and immunophenotyping of human monocytes during HIF-prolyl hydroxylase inhibition.a-e, FcγR expression levels and FcγR A:I (FcγR activating:inhibitory) ratio were quantified using flow cytometry (n = 7).Statistical significance between groups was assessed using a paired two-tailed Wilcoxon test (p < 0.05, p < 0.01 and p < 0.001).f-l, RNA-Seq analysis of the transcriptome of untreated and DMOG treated monocytes cultured for 0 (fresh), 2, 10, and 24 h.f, Monocyte gene expression time course trajectories in principal component space (dimensions 1 and 2).Principal Component Analysis (PCA) on 6198 differentially expressed genes for untreated versus DMOG-treated comparisons.g, Pre-ranked GSEA; genes were ranked according to their differential expression between monocytes at 10 h post-DMOG treatment and fresh monocytes.Twenty-five Hallmark gene sets (v7.2) were significantly overrepresented (FDR < 0.05), with gene sets of interest highlighted red, indicating upregulated gene expression across all time-points, and the oxidative phosphorylation gene set highlighted blue, showing downregulation of gene expression.h Expression fold changes (log2(FC)) for FCGR genes between untreated and DMOG-treated monocytes.i, Enrichment plot of Winter hypoxia gene set in monocytes at 10 h post-DMOG treatment vs fresh monocytes (NES = 2.33).j, Enrichment plot of the Hallmark hypoxia gene set in DMOG treated monocytes at 10 h vs fresh monocytes (NES = 2.86).k, Gene expression heat map for differentially expressed genes of interest in untreated (U) and DMOG-treated monocytes at 10 h post-culture.Columns represent monocyte samples from 7 donors.l, Upstream regulator analysis was performed using IPA.Heat map of activation z-scores indicates the top 50 transcription factor genes and proteins predicted to be activating or inhibiting when comparing untreated monocytes with DMOG treated monocytes (n = 7 per group).Also see Additional file 3: Fig. S3 (See figure on next page.)genes, revealing that multiple hypoxia pathway genes were evident, with HIF1A, HIF2A (EPAS1), and ARNT (HIF1B) present in the top 10.Furthermore, JUN and FOS which encode proteins that form the AP-1 transcription factor complex, were also identified within the top 50 (Fig. 3i).GSEA revealed a hypoxia gene signature that was detectable at 2 h and sustained through to the 24 h time period post-DMOG treatment (Additional file 3: Fig. S3d).HIF1A and HIF2A were amongst the top 10 upstream regulators of differentially expressed genes at both the 10-and 24-h time points post-treatment with DMOG (Additional file 3: Fig. S3e). We next profiled chromatin accessibility in untreated and DMOG-treated primary human monocytes using ATAC-seq (using material from the same experiment reported in Fig. 3).Chromatin accessibility analysis of DNA from 24 h post-treatment, revealed considerable separation, when comparing differentially open and closed regions of the genome between untreated and DMOG treated monocytes as viewed by PCA (Fig. 4a).Furthermore, hierarchical clustering analysis of significantly differentially open and closed regions revealed marked dissimilarity between donor matched untreated and DMOG treated monocytes (Fig. 4b).Canonical transcription factor DNA binding motifs were next quantified within significantly opened regions in DMOG treated samples when compared to untreated samples.HIF-1α, HIF-2α and proteins which form the AP-1 transcription factor complex were amongst the top 50 transcription factors predicted to access more open regions of the genome after DMOG treatment (Fig. 4c).Amongst the most significantly open genomic regions in DMOG treated monocytes when compared to donor matched untreated monocytes, was the promotor region of EGNL3, which encodes PHD2 and is known to be upregulated in response to HIF-α protein stabilization (Fig. 4d).We also looked for DMOG-induced changes directly in the low affinity FCGR locus.FCGR2B and FCGR2C share sequence homology resulting in considerable multimapping for these two genes.Nonetheless, when comparing both multi-mapped and uniquely-mapped reads for FCGR2B we observed no significantly open genomic regions (peaks) between untreated and DMOG treated monocytes (Fig. 4e).Furthermore, visualization of the multi-mapped 1 Kb region upstream of the FCGR2B gene transcription start site (TSS), did not reveal additional or more pronounced peaks (Fig. 4f ).In contrast, additional and pronounced peaks were observed for EGLN3 (which was one of the most significantly open genes in DMOG treated monocytes; Fig. 4g).This indicated that the enhancement of FCGR2B gene expression in DMOG treated monocytes was not mediated by increased chromatin accessibility within the FCGR2B gene locus, but instead was likely driven by altered transcription factor binding.Open peaks were scanned for transcription factor binding sites using the ORegAnno database, in differentially regulated regions of the genome when comparing untreated versus DMOG-treated monocytes.This analysis revealed a non-random distribution for HIF-1α and HIF-2α binding motifs, in contrast to GATA-binding factor 2 and GATA-binding factor 3 binding motifs which were randomly distributed in open regions of the genome (Fig. 4h).To determine whether HIFs and AP-1 could directly interact with the FCGR2B gene promotor, we first searched for the HIF-1β/HIF-α and AP-1 canonical core motifs (as defined by the JASPAR open-access database for TF binding profiles, Additional file 4: Fig. S4a), in the 1 Kb region upstream of the FCGR2B gene TSS, however, precise matches were not located within this region.Olferiev et al., previously described a non-canonical motif for AP-1 in the FCGR2B gene promotor [69] and we also located this motif at position -339 upstream of the TSS.Additionally, we also identified a non-canonical hypoxia response element (HRE) and a potential HIF binding motif at position -835 (Additional file 4: Fig. S4b).Similar non-canonical HIF binding motifs have been previously reported for CD73 [83] and PEPCK [84].To confirm whether these molecules were enriched for binding to the FCGR2B gene promotor during DMOG treatment we performed ChIP.Using specific mAb for c-Jun, HIF-1α, and HIF-2α we performed ChIP-quantitative PCR analysis on mAb extracted DNA to detect the FCGR2B promotor region (normalized with the negative isotype control mAb) revealing that only c-Jun and HIF-2a increasingly interact directly with the FCGR2B gene promotor region at 24 h post-DMOG treatment (Fig. 4i).Additionally, we also analyzed ChIP-Seq data generated by Tausendschön et al., who utilized HIF-1α and HIF-2α specific mAbs, to determine genomic HIF-α interactions in human MDMs cultured at 1% O 2 for 8 h [60].At this earlier time point (our ChIP assay was carried out using monocytes cultured for 24 h), a peak within the 10 Kb region upstream of the FCGR2B gene was detected in both anti-HIF-1α and HIF-2α 'ChIPed' DNA of hypoxic but not normoxic MDMs, but no HIF-α interaction was detected in the 1 Kb region upstream of the FCGR2B gene TSS (Additional file 4: Fig. S4c).Tausendschön et al., also knocked down HIF1A and HIF2A genes in normoxic and hypoxic human MDMs using siRNA [60] and our analysis of this data also revealed downregulation of FCGR2B expression, particularly following HIF2A knockdown in hypoxic MDMs (Additional file 4: Fig. S4d).Altogether these data show that DMOG treatment of human monocytes potently induces a hypoxia related gene signature alongside prominent transcriptional modulation by HIFs and AP-1, that is coincident with an enhancement of FcγRIIb expression and consequent downregulation of the FcγR A:I ratio. ", "section_name": "HIF-prolyl hydroxylase (HIF-PH) inhibition induces FcγRIIb upregulation on human monocytes", "section_num": null }, { "section_content": "To confirm that hypoxic conditions upregulate FcγRIIb on mononuclear phagocytes, we cultured these cells under physiological hypoxia.Human LD monocytes cultured under hypoxic conditions (1% or 3% O 2 ) significantly upregulated FcγRIIb when compared to monocytes cultured under normoxic conditions (21% O 2 ), (p < 0.01, Fig. 5a-b).Treatment of PBMCs with the pan-HIF-PH inhibitor, DMOG, or the prolyl hydroxylase domain 2 (PHD2) inhibitor, Roxadustat [85], led to comparable enhancement of FcγRIIb expression (p < 0.0001 for both inhibitors, Fig. 5c-d).DMOG treatment of monocytic THP-1 cells also enhanced FcγRIIb expression in a dose dependent manner (Fig. 5e).Equivalent experiments with various B cell lines saw no change in FcγRIIb expression (data not shown), again underlining differential regulation in B versus myeloid cells.We next differentiated monocytes into macrophages using M-CSF over 7 days and then stimulated them with LPS/IFN-γ (M1), IL4/IL-13 (M2) or left them untreated (M0) for 2 days in the absence or presence of DMOG.DMOG treatment of all three types of MDMs significantly upregulated FcγRIIb, being particularly evident for M1 and M2 macrophages (p < 0.001 and p < 0.01 for M1 and M2, respectively, Fig. 5f ) and confirmed by Western blot (Fig. 5g).Importantly, DMOG treatment of human monocytes, MDMs and THP-1 cells consistently and significantly decreased the FcγR A:I ratio (Fig. 5h), indicating that HIF activation can profoundly alter FcγR expression on mononuclear phagocytes in a manner that may be detrimental to mAb immunotherapy. ", "section_name": "Physiological hypoxia and pharmacological HIF activation lead to comparable enhancement of FcγRIIb expression levels", "section_num": null }, { "section_content": "To further define the mechanism underlying FCGR2B upregulation and ascertain the importance of AP-1 and HIFs in enhancing FcγRIIb cell surface expression on mononuclear phagocytes, we first used a series of small molecule inhibitors.Digoxin has been reported to inhibit HIF-1α translation [86,87] and we observed significant inhibition of FcγRIIb upregulation (p < 0.0001) and changes in FcγR A:I ratio (p < 0.05) on HD monocytes following treatment with Digoxin (Additional file 5: Fig. S5a-b), supporting a role for HIFs.Furthermore, treatment of LD monocytes with the VHL inhibitor; VH298 [38], which stabilizes HIF-α subunit protein expression, increased FcγRIIb expression (p < 0.01) and decreased the FcγR A:I ratio (p < 0.05, Fig. 5i).Simultaneous treatment of DMOG treated-monocytes with the HIF-α inhibitor FM19G11 [39], diminished the increase in FcγRIIb expression (p < 0.01) and consequently increased the FcγR A:I ratio (p < 0.05, Fig. 5j).Significant upregulation of c-Jun protein in DMOG-treated monocytes was confirmed by Western Blot (Fig. 5k-l).Furthermore, culturing DMOG-treated monocytes with a c-Jun peptide inhibitor, which abrogates JNK/c-Jun interactions [40], also led to a potent inhibition of FcγRIIb upregulation, impairing the reduction in FcγR A:I ratio, (Fig. 5m-n).M1 MDMs treated with the c-Jun peptide inhibitor also experienced a similar impairment of change of FcγR expression levels, in response to DMOG treatment (Additional file 5: Fig. S5c-d).These findings indicated that AP-1 and HIFs were involved in the enhancement of FcγRIIb expression on hypoxic mononuclear phagocytes. However, to more precisely assess their contribution, we used siRNA-mediated knock-down in untreated and DMOG treated monocytes.We first knocked down HIF1A in LD, HD and DMOG treated human monocytes using HIF1A specific siRNA, confirming the knock down by measuring HIF-1α expression via Western blot (Additional file 5: Fig. S5e).Although ChIP assessment revealed that HIF-1α did not interact with FCGR2B gene promotor at 24 h post-DMOG treatment (Fig. 4i), here HIF1A knock-down inhibited FcγRIIb upregulation in both HD (Additional file 5: Fig. S5f ) and DMOG treated monocytes (Additional file 5: Fig. S5g), confirmed by measuring FcγRIIb expression using flow cytometry.This indicated a non-redundant role for HIF-1α in enhancing FcγRIIb expression on mononuclear phagocytes under hypoxia-like conditions.Next we knocked down HIF2A and JUN using siRNA, which almost entirely prevented the upregulation of FcγRIIb in DMOG treated monocytes (Fig. 5o-p) and consequently prevented the reduction in the FcγR A:I ratio (Fig. 5q), when assessed by flow cytometry.HIF2A and JUN knock downs using these siRNA were confirmed by assessing HIF-2α and c-Jun by Western blot (Additional file 5: Fig. S5h).Prevention of FcγRIIb upregulation in DMOG treated monocytes post-HIF2A and JUN knockdowns were also confirmed by Western blot (Fig. 5r-s).AP-1 activity can be regulated by post-translational modification, including phosphorylation by the mitogen-activated protein kinase (MAPK) family which comprises of MAPKs, the extracellular signal regulated kinase (ERK), p38 MAPK and c-Jun NH2-terminal kinase (JNK) [88].Therefore, we determined the phosphorylation status of c-Fos (p-c-Fos) in DMOG treated monocytes and observed that it was elevated relative to untreated monocytes (Additional file 5: Fig. S5i), indicating the association of AP-1 activation with HIF-α protein stabilization.These observations led us to conclude that the enhancement of FcγRIIb expression following HIF activation in mononuclear phagocytes is dependent upon the protein expression and activation of HIF-1α, HIF-2α and AP-1, all of which potentially directly interact with the FCGR2B gene loci upstream of its TSS. ", "section_name": "Upregulation of FcγRIIb in human mononuclear phagocytes is mediated by HIFs and AP-1", "section_num": null }, { "section_content": "To explore the broader relevance of our observations regarding the hypoxia-induced upregulation of FcγRIIb expression on mononuclear phagocytes, we immunophenotyped these cell types in contexts where hypoxia is likely present such as within human and murine tumors, and tumor associated ascites.First, we compared FcγRIIb expression on peripheral blood and pleural fluid monocytes from mesothelioma patients where the oxygen levels would be expected to differ [89].Monocytes in the pleural fluid of these patients expressed significantly elevated levels of FcγRIIb relative to donor matched peripheral blood monocytes (p < 0.01, Fig. 6a-b).Pleural fluid monocytes also possessed a significantly lower FcγR A:I ratio in comparison to peripheral blood monocytes (p < 0.0001, Fig. 6b).Pleural fluid and peripheral blood neutrophils were negative for FcγRIIb and B cells expressed similar levels of FcγRIIb in both niches (data not shown).FcγRIIb bright monocytes were also detected in the ascites of breast cancer patients (Fig. 6c).In renal cell carcinoma (RCC), a tumor type associated with high HIF-α expression [90], both monocytes and macrophages expressed significantly elevated levels of FcγRIIb, relative to donor matched counterparts in healthy kidney tissue (p < 0.05, Fig. 6d-e).When comparing splenic and tumor FcγRII expression in WT C57BL/6 J mice, expression was elevated in all three subcutaneous tumor models we examined (MCA205, CT26 and EG7).FcγRII expression was significantly elevated on CD11b + Ly6C hi monocytes, in mice inoculated with the MCA205 (p < 0.01), CT26 (p < 0.05) and EG7 subcutaneous tumors (p < 0.0001), consequently reducing FcγR A:I ratios on monocytes in CT26 (p < 0.05) and EG7 (p < 0.0001) tumors relative to matched spleens (Fig. 6g).Tumor associated F4/80 + macrophages in mice inoculated with the MCA205 (p < 0.01), CT26 (p = 0.062) and EG7 (p < 0.0001) also expressed elevated levels of FcγRII when compared to matched splenic F4/80 + cells, with the FcγR A:I ratio similarly and significantly reduced in MCA205 (p < 0.01), CT26 (p < 0.01) and EG7 (p < 0.001) tumors (Fig. 6h).To investigate the expression of FcγRII in hypoxic regions of tumors we utilized immunofluorescence microscopy.Hypoxic regions of tumor sections were identified using Hypoxyprobe (pimonidazole) and co-localization with FcγRII was assessed.These studies revealed FcγRII expression was concurrent with hypoxic regions of the tumor within three different tumor models: CT26 tumors (Fig. 6i), MC38 and EO771 (Additional file 6: Fig. S6a-b).These observations indicate that FcγRIIb expression on mononuclear phagocytes is elevated when they are associated with, or resident within, human and murine tumors, where it profoundly impacts the FcγR A:I ratio.We hypothesized that the FcγRIIb bright phenotype of these (See figure on next page.)Fig. 6 FcγR expression on tumor associated mononuclear phagocytes.a, Representative histograms showing FcγR expression on fresh donor matched peripheral blood (PB) and pleural fluid (PF) monocytes from a single mesothelioma patient.b, FcγRIIb expression (left) and FcγR A:I ratio (right) were quantified for PB and PF monocytes (FSC hi CD45 + CD14 + cells) sourced from mesothelioma patients using flow cytometry (n = 6 per group).c, Representative histograms showing expression of FcγRIIb on fresh monocytes isolated from lymphocele taken from 3 breast cancer patients.d, Representative histograms showing FcγRIIb expression on fresh donor matched renal monocytes (FSChiCD45+CD14+ cells) and macrophages (FSChiCD45+CD163+ cells) in normal kidney tissue and tumor from a single renal cell carcinoma (RCC) patient.e, FcγRIIb expression (left) and FcγR A:I ratio (right) were quantified for monocytes and macrophages sourced from normal kidney tissue and donor matched RCC specimens using flow cytometry (n = 5 per group).f, Representative histograms showing FcγRIIb expression on splenic and MCA205, CT26 and EG7 tumor associated CD11b+F4/80+ macrophages.g, Comparison of murine FcγRII expression (left) and FcγR A:I ratio (right) on CD11b+Ly6C+ monocytes and h, CD11blo/F4/80+ macrophages in recipient matched spleen and subcutaneous MCA205, CT26 and EG7 tumors (n = 5-9 per group).Each point on the graphs represents a unique human subject or mouse and bars represent group means.Statistical significance between groups was assessed using a paired two-tailed Wilcoxon test (p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 and ns = non-significant).i, Immunofluorescence staining of hypoxic regions using hypoxyprobe (hypoxia probe) and anti-mouse FcγRII, on sections taken from a CT26 tumor.Localization of FcγRII expression in hypoxic regions is shown.Images representative of stained sections from 5 different mice.Scale bars 100 μm.Also see Additional file 6: Fig. S6 tumor associated mononuclear phagocytes had the potential to impair direct targeting mAb immunotherapy. ", "section_name": "Tumor-associated human and murine mononuclear phagocytes are FcγRIIb bright", "section_num": null }, { "section_content": "We next sought to determine the functional consequence of FcγRIIb upregulation on mononuclear phagocytes.To investigate this, we assessed the ability of HD versus LD human monocytes to phagocytose RBCs opsonized with anti-D mAb.We observed that the phagocytic function of HD monocytes was significantly diminished in comparison to LD monocytes (p < 0.05, Fig. 7a andb).When FcγRIIb on HD monocytes was blocked using a F(ab') 2 FcγRIIb specific antibody, phagocytic function was significantly improved (p < 0.05) unlike when the activating FcγRIIa was blocked (Fig. 7a-b). Next, we examined the impact of hypoxia on MDMs and used untreated and HIF-PHD inhibitor-treated M0, M1 and M2 MDMs as effector cells in ADCP assays.We observed phagocytosis of Rituximab opsonized CLL cells was significantly decreased by DMOG or roxadustat treatment in M0, M1 and M2 MDMs (Fig. 7c-e).This significant reduction in ADCP function of DMOGtreated MDMs was also observed when CLL cells were opsonized with another anti-CD20 mAb, obinutuzumab (Fig. 7f ). Having established these significant effects in vitro, we next explored the effects of hypoxia induction, using HIF-PH inhibition, on FcγR expression and target cell depletion in vivo.In wild type C57BL/6 J mice, DMOG introduction into the peritoneum significantly increased FcγRII expression on macrophages (Fig. 7g) and monocytes (Additional file 7: Fig. S7a-b).Similar effects were seen with Roxadustat (Additional file 7: Fig. S7c-d).Furthermore, HIF-PH inhibitor treatment also enhanced FcγRII expression on peripheral blood monocytes and on splenic monocytes and macrophages in wild type C57BL/6 J mice (Additional file 7: Fig. S7e-g). Having established the ability of these HIF-PH inhibitors to mediate in vivo changes in FcγR expression and A:I ratio, we assessed the impact of these changes on mAb-mediated target cell deletion.Accordingly, Roxadustat was administered to C57BL/6 J mice before treatment with the potent anti-mCD20 mAb; 18B12 [36] and B cell deletion was assessed.Roxadustat evoked a significant impairment in B cell deletion (Additional file 7: Fig. S7h).Furthermore, Rituximab mediated depletion of human CD20 + (hCD20 + ) EL4 tumor cells in the peritoneum of roxadustat treated C57BL/6 J mice was also significantly impaired (p < 0.05, additional file 7: Fig. S7i-j).To extend the translational relevance of our findings, we next assessed the effects of HIF-PH inhibition on human FcγRIIb (hFcγRIIb) expression and mAb mediated target depletion in transgenic mice expressing the human FCGR2B and CD20 genes and lacking the murine FcγRII (hFcγRIIb +/-x mFcγRII -/-x hCD20 +/-mice).DMOG treatment in these mice resulted in significant increases of hFcγRIIb expression on monocytes, macrophages and neutrophils in the spleen (Fig. 7h).Significant decreases in the FcγR A:I ratio, because of DMOG mediated enhancement of FcγRIIb expression, were also observed in splenic monocytes and macrophages (Fig. 7i).Rituximab-mediated splenic B cell depletion was significantly impaired in DMOG-treated transgenic hFcγRIIb +/-x mFcγRII -/-x hCD20 +/-mice (Fig. 7j).Fig. 7 The impact of hypoxia-driven FcγRIIb upregulation on mAb mediated target cell depletion.a, Flow cytometry plots showing levels of uptake of CSFE + red blood cells (RBCs) by LD and HD monocytes.RBCs sourced from Rhesus D + individuals were opsonised with control cetuximab (CTX) or anti-Rhesus D antigen specific mAb (αD).RBCs used as targets for LD and HD pre-cultured monocytes pre-treated with or without anti-FcγRIIb (αFcγRIIb) blocking mAb.b, RBC phagocytosis quantified for 6 donors.c, Flow cytometry plots showing Rituximab mediated uptake of CLL cells by FcγRIIIa + M1 macrophages generated with or without DMOG.d, CLL cells opsonised with Rituximab and cultured with M0, M1 or M2 MDMs generated in the absence or presence of DMOG or e, Roxadustat and the percentage of phagocytic MDMs were determined by flow cytometry (n = 6-8 per group).f, Phagocytosis of CLL cells mediated by Obinutuzumab (n = 6 per group).g, FcγR expression on F4/80 + macrophages in the peritoneal lavage of WT C57BL/6 mice treated with DMOG or PBS control.i.p., determined using flow cytometry (n = 6 per group).h, FcγRIIb expression levels and i, FcγR A:I ratio were determined by flow cytometry in splenic monocytes (Mono), macrophages (Mac) and granulocytes (Gran) of DMOG or PBS treated hFcγRIIb/mFcγRIIKO mice (n = 8 per group).j, hFcγRIIb/mFcγRIIKO/hCD20 mice were treated with DMOG or vehicle PBS control i.p. for 72 h prior to receiving Rituximab (RTX) or CTX isotype control.%CD19 + cells in the peripheral blood of each mouse were determined using flow cytometry (n = 8-10 per group).k, hFcγRIIb/mFcγRIIKO mice were treated with DMOG or PBS i.p. for 72 h prior to receiving CFSE labelled target splenocytes from hCD20/mFcγRIIKO mice and non-target splenocytes from WT C57BL/6 mice, i.v.These mice were treated with DMOG or PBS i.p. prior to receiving RTX or CTX 24 h later.Flow cytometry plots are shown for the depletion of target and non-target splenocytes, and l, data is presented as CD19 + cell target:non-target ratio (n = 5 per group).m, Flow cytometry plots showing hFcγRIIb expression on liver and n, peritoneal lavage F4/80 + macrophages 72 h post-treatment with DMOG or PBS control, i.p., in hFcγRIIb/mFcγRIIKO mice, and o, quantified for 5 mice per group.p, hFcγRIIb/mFcγRIIKO mice were treated with DMOG or PBS i.p. for 60 h prior to receiving CFSE labelled EL4-huCD20 + cells following treatment with RTX or CTX, and DMOG or PBS.Histograms showing depletion of target EL4-huCD20 + cells in the peritoneum by RTX in the absence and presence of DMOG and q, EL4-huCD20 + cell depletion in the peritoneal lavage quantified using flow cytometry.Bars represent group means.Statistical significance was assessed using an unpaired two-tailed t-test, a paired two-tailed Wilcoxon test, or a one-way ANOVA for the in vivo cell depletion experiments (p < 0.05, p < 0.01, p < 0.001, ***p < 0.0001 and ns = non-significant).Also see Additional file 7: Fig. S7 (See figure on next page.) To refine this in vivo model, we adoptively transferred wild type (non-target) or transgenic hCD20 + splenocytes (target) into hFcγRIIb +/-x mFcγRII -/-mice.We observed rituximab-mediated depletion of the huCD20 + B cells was impaired post-DMOG treatment, whereas the non-target wild type B cell frequencies remained constant across all treatment groups (p < 0.05, Fig. 7k-l).Moreover, hFcγRIIb expression on liver macrophages was significantly elevated in DMOG treated hFcγRIIb +/-x mFcγRII -/-mice (p < 0.05) with a similar trend for peritoneal macrophages (p = 0.06, Fig. 7m-o).Finally, we assessed rituximab mediated depletion of malignant hCD20 + EL4 tumor cells from the peritoneum of hFcγRIIb +/-x mFcγRII -/-mice.We observed that DMOG treatment also significantly impaired target cell depletion in this model (p < 0.05, Fig. 7p-q).In summary, HIF activation via HD culture or HIF-PHD inhibition significantly impairs the ability of monocytes and macrophages to phagocytose and deplete mAbopsonized cellular targets in vitro and diminishes direct targeting anti-cancer mAb therapy in vivo. ", "section_name": "mAb-mediated phagocytic function is impaired in FcγRIIb bright mononuclear phagocytes", "section_num": null }, { "section_content": "We demonstrate that exposure to physiological or pharmacological hypoxia induces rapid upregulation of the inhibitory IgG Fc receptor, FcγRIIb, on mononuclear phagocytes.This enhancement of FcγRIIb expression, diminishes the FcγR A:I ratio, consequently impairing the ability of monocytes and macrophages to phagocytose mAb opsonized cancer cells and cellular targets.The generation of these 'FcγRIIb bright ' mononuclear phagocytes under hypoxic conditions is transcriptionally driven and is dependent upon AP-1, as well as HIF-1α and HIF-2α interactions with the FCGR2B gene promotor region.Detection of FcγRIIb bright mononuclear phagocytes resident within tumors or in associated niches asserts that these cells may be crucial determinants in reducing the efficacy of widely used direct-targeting mAbs.Our findings highlight a novel mononuclear phagocyte phenotype that in addition to being fostered by the hypoxic TME may be actively selected in rapidly growing solid malignancies thereby diminishing the efficacy of mAb immunotherapies. We observed that under HD conditions or HIF-PH inhibition, human monocytes rapidly upregulate FcγRIIb, acquiring an FcγRIIb bright phenotype, to display levels exceeding other abundantly expressed surface antigens such as MHC Class I. Furthermore, monocytes obtained from RCC patients or tumor associated niches, such as in the pleural cavity of mesothelioma patients or breast cancer patient ascites, also possess an FcγRIIb bright phenotype, contending that this phenotype is physiologically relevant.In vitro, we primarily modelled the effects of hypoxia on human monocytes, using HD cell culture (in which O 2 levels rapidly drop to as low as 0.1%) and treatment with the HIF-α protein stabilising reagent; DMOG.It has previously been shown that there is a high degree of concordance between HIF-α binding in human proximal tubular epithelial HKC-8 cells exposed to DMOG and those cultured at 1% O 2, where both stimuli produce comparable genome-wide patterns of HIF DNA-binding [91].GSEA of HD and DMOG treated monocyte transcriptomes also revealed excellent concordance with hypoxia gene signatures, that were amongst the most prominent and coincident with the upregulation of FCGR2B expression. Similar hypoxia-correlated FcγRIIb upregulation was also seen in macrophages and TAMs, which holds further translational significance, as these cells are the key effector mononuclear phagocyte populations with respect to therapeutic mAb-mediated elimination of cancer cells [22,93,94].Macrophages abundantly infiltrate tumors and are found in normoxic and hypoxic tumor compartments, albeit in different polarization states [95].We observed elevated FcγRIIb expression on macrophages in human RCC and 4 different syngeneic murine subcutaneous tumors spanning colorectal, fibrosarcoma, thymoma and breast cancer models, relative to matched splenic macrophage FcγRIIb expression.Furthermore, HIF-PH inhibitor treatment of WT or hFcγRIIb Tg mice upregulated FcγRIIb on mononuclear phagocytes in vivo.We propose that this FcγRIIb bright phenotype may represent a key determinant of resistance to mAb therapy in the TME.However, hypoxia alone is unlikely to be the only stimulus influencing macrophage behaviour within the TME [96], and the integrated effects of hypoxia, cytokines and multiple other interactions will ultimately shape macrophage phenotype and function.Indeed, hypoxia is a common feature in many pathophysiological states [97][98][99] in which the respective macrophage phenotype might differ.For instance, whereas hypoxic TAMs are more immunosuppressive, TLR-signalling in sepsis might be expected to induce strong cellular activation even in the presence of hypoxia [17,100].Nonetheless, at least with MDMs we observed that DMOG treatment upregulated FcγRIIb on all three types of macrophage polarisation states (M0, M1 and M2) we examined, perhaps indicating that hypoxia may have a powerful and pervasive diminishing effect on FcγR A:I ratio and therefore ADCP. HIF1A, HIF2A and JUN gene knockdowns revealed that both HIF-α subunits and c-Jun have roles in mediating hypoxia-mediated FCGR2B gene expression on mononuclear phagocytes.This data is supported by observations in HeLa cells following exposure to hypoxia [101] where AP-1 transcriptional activity is increased, and AP-1 and HIF-1α binding is required in close proximity for the induction of up to ~ 20% of the HIF binding sites in hypoxic human MDMs [60].Olferiev et al., have previously reported that AP-1 family members bind to the FCGR2B promoter in PMA/ionomycin activated CL-01 and U937 cells [69].Using ChIP assays followed by PCR amplification, we also observed that c-Jun interacted with the FCGR2B gene promotor region containing the non-canonical AP-1 motif; TGC ATC A (at -345 upstream of the TSS), in DMOG-treated monocytes.The interaction of AP-1 transcription factors with another non-canonical motif; TGC GTC A contained in the HLA-DR gene promoter in a B-cell lymphoma line, provides a further example that AP-1 is capable of interacting with non-canonical consensus DNA sequences and inducing gene expression [102].We also observed that DMOGtreated monocytes express higher levels of c-Jun protein and RNA-Seq analysis revealed that expression of AP-1 components, JUN and FOS, also increases post-DMOG treatment. We further investigated whether HIFs themselves induce FCGR2B transcription.In MCF-7 human breast cancer cells, both HIF-1α and HIF-2α primarily bind relatively GC rich DNAse1 sensitive genomic regions, reflecting the concentration of hypoxia response elements (HRE) within chromatin accessible promoter regions and over 500 such HIF-binding sites have been identified across the human genome [92,103].HIFs primarily mediate gene expression by binding to HREs, a gene sequence which contains the RCGTG core motif (with preference of A over G at the R position), beyond which a preference is also observed for a CAC motif [91].We sought to determine whether the FCGR2B promotor region contains HREs and three ACGTC and six GCGTC motifs within the 15 Kb region upstream of the FCGR2B TSS were identified (data not shown).Furthermore, our analysis, of publicly available ChIP-seq data, sequencing HIF-1α and HIF-2α bound DNA from normoxic and hypoxic human MDMs [60], revealed HIF-α interaction at distal regions > 10 Kb upstream of the TSS at 8 h posthypoxia (Additional file 4: Fig. S4c).However, we identified that the nearest canonical HRE (GCGTG) motif to the FCGR2B TSS is at position -3916 upstream of the TSS.Moreover, using ChIP assays we also identified a sequence close to the AP-1 binding site (at position -838 upstream of the TSS) to be a potential non-canonical HRE with which HIF-2α (but not HIF-1α) may interact in DMOG-treated monocytes.This motif is a non-canonical CCGTG sequence, which has been previously described for CD73 [83] and PEPCK [84] and additionally a CAC motif is also located in close proximity to this motif (Additional file 4: Fig. S4b).Although a role for HIF-2α in the regulation of FCGR2B expression was ascertained by ChIP, HIF1A gene knock down also revealed its nonredundant role.It has previously been reported that in murine embryonic fibroblasts initial exposure to hypoxia stimulates expression of c-Jun and transient activation of protein kinase and phosphatase activities that regulate c-Jun/AP-1 activity dependent upon HIF1-α [104].Evidence for direct cooperation between AP-1 and HIF-1α has been reported for VEGF and TH which contain functional AP-1 and HRE sites [105,106].Here we propose a mechanism by which AP-1, HIF-1α/HIF-1β and HIF-2α/ HIF-1β transcription factor complexes cooperate to mediate marked cell surface FcγRIIB upregulation under hypoxic conditions on human mononuclear phagocytes. Previous studies have shown that monocytes and macrophages are key mediators of cancer cell depletion in therapeutic settings utilising direct-targeting mAbs such as Rituximab, Cetuximab and Herceptin [107][108][109][110]. Uchida et al., have demonstrated that anti-CD20 mAb mediated depletion of circulating B cells in mice was dependent upon activating FcγR since B cell depletion was almost entirely lost in FcR common γ-chain-null mice (that lack activating FcγRI, FcγRIII and FcγRIV) and monocytes were identified as the key effector population in this context [20], which we and others confirmed in later studies [21].In the current study we report that Rituximab meditated ADCP of CLL cells by human MDMs is compromised by HIF-PH inhibitor treatment and that the same treatment compromises anti-CD20 mAb mediated depletion of cell targets in multiple niches in vivo.We attribute these outcomes to the potency of hypoxia-mediated upregulation of mononuclear phagocyte FcγRIIb. Although inhibitory for direct targeting mAb as indicated, it is likely that these hypoxia-mediated changes in FcγR are not detrimental in all scenarios.For example, FcγRIIb is known to act as a positive regulator of several agonistic mAbs targeting immune receptors such as CD40, OX40, 4-1BB and CD28 by providing higher levels of receptor cross-linking [32,[111][112][113].Therefore, hypoxia-mediated upregulation of FcγRIIb in the TME may even serve as an important component of efficacy for these mAb. Further studies are needed to determine if hypoxia could serve as a prognostic marker for response to different mAb therapies (negatively regulating direct targeting modalities but augmenting agonistic immunomodulatory mAb).Similarly, whether hypoxia can be appropriately modulated to improve such therapies remains to be demonstrated.In clinical settings the lack of an accurate and approved method to evaluate tumor hypoxia accounts for the limited capacity to intervene with a personalized hypoxia-based therapy.HIF-1α is a well-appreciated target for cancer therapies, and drugs that indirectly inhibit hypoxia/HIF-1α signalling such as digoxin and acriflavine, have been reported to have relevant impacts -for example decreasing lung metastasis in an orthotopic breast cancer model [114].However, efforts to develop highly specific and efficacious small molecule HIF-1α inhibitors have been largely unsuccessful [115].Nevertheless, alternative methods to modify hypoxic regions within tumors include supplemental oxygen, anti-VEGF therapy and use of the chemotherapeutic reagent, and hypoxia activated prodrug; evofosfamide [116][117][118][119], which could all be explored in the context of mAb therapy.Similarly, mAb-mediated blockade of the hypoxia upregulated FcγRIIb on TAMs is an exciting and emerging strategy [30,120,121], with demonstration of combination effects with several direct targeting mAb in preclinical models and encouraging recent evidence in the clinic (Jerkeman et al., 2020, article accepted and in press, https:// doi.org/ 10. 1182/ blood-2020-140219). It will be important to understand what degree of the tumor hypoxia effects on myeloid (and other) cells can be overcome by blockade of Fc:FcγRIIb interactions, potentially leading to additional TME O 2 modifying approaches as indicated above.Targeting the tumor myeloid landscape and specifically the FcγRIIb bright phenotype in combination with established directtargeting mAbs provides a potentially powerful novel strategy to overcome disease resistance to current and evolving antibody immunotherapies. ", "section_name": "Discussion", "section_num": null } ]	[ { "section_content": "We would like to thank the members of the Antibody and Vaccine group for useful discussions and the pre-clinical unit staff for animal husbandry and assistance.ATAC-seq samples were prepared by Victoria Gernedl in the Bock lab and bioinformatically processed by Bekir Ergüner.The sequencing was performed by the Biomedical Sequencing Facility at the Center for Molecular Medicine, Vienna.We would like to thank Dr. Francesco Forconi, Dr. N Kathleen Potter, Dr. Ian Tracy and Mrs. Isla Henderson (University of Southampton, The Human Tissue Bank) for provision of CLL samples.We would like to thank Ms. Deborah Donovan (Southampton NHS Trust) for assisting us in the use of the ABL 835 FLEX analyzer, in order to measure metabolites and electrolytes in cell culture supernatants.We would like to thank Prof J and D Mann (University of Newcastle) for insightful initial discussions.We would like to thank Dr Zoë S Walters and Dr Ian Tracy for their expertise on ChIP assays.We would like to thank CRUK Southampton Centre Bioinformatics core facility for supporting this work.Finally, we would like to acknowledge Dr Charles Tilley and Dr Janlyn Falconer for assistance with pathology and provision of primary tumor clinical samples, respectively. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Microarray and sequencing data generated in this study are deposited in the Gene Expression Omnibus under the following accession numbers: GSE165643 (Microarray for HD monocytes and B-cells), GSE166100 (ATACseq for LD-HD monocytes) and GSE165999 (RNA-seq (GSE165998) and ATAC-seq (GSE165997) for DMOG time course monocytes). ", "section_name": "Availability of supporting data", "section_num": null }, { "section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s13046-022-02294-5. Authors' contributions K.H. designed and performed the experiments, analyzed and interpreted data and wrote the manuscript.R.L., R.C.G.S., K.T.J.M., M.G., S.M., K.L.S.C., R.J.O., R.B.F., S.J., S.G.B., T.M., L.N.D., C.E.H., R.S.K., J.L., J.D., H.M., S.J.C., R.J.S., M.J.C., C.H.O., R.I.C., R.R.F. and S.M.T. generated and provided key reagents, performed experiments, wrote specific method sections and analyzed data.A.R., B.F. and M.J.G. discussed and interpreted data and edited the manuscript, J.C.S., S.M.T., S.A.B. and M.S.C. acquired funding, designed the study, supervised data collection, discussed and interpreted data and edited the manuscript.The author(s) read and approved the final manuscript. Anonymized leukocyte cones were sourced from healthy adult donors attending blood donation clinics at the National Blood Service (Southampton, UK).The use of leukocyte cones for this work was approved by the University of Southampton Faculty of Medicine Ethics Committee and the East of Scotland Research Ethics Service, Tayside, UK, Research ethical committee (REC) reference number: 16/ES/0048.Clinical samples from 6 anonymized mesothelioma patients (REC reference number: 13/SW/0128) and Donor matched Renal cell carcinoma (RCC) and non-cancerous healthy kidney tissue samples were obtained from resected kidneys from 5 RCC patients (REC reference number: 17/WA/0241).Lymphocele samples were sourced from 3 anonymized breast cancer patents (REC reference number: 10/H0504/73, for breast cancer patient samples).Peripheral blood samples were taken from Chronic Lymphoblastic Leukemia (CLL) patients and anonymized before experimental use (REC reference number: 10/H0504/187).These aforementioned clinical samples were released from the Human Tissue Authority Licensed University of Southampton, Cancer Sciences Tissue Bank, as approved by the Southampton and South West Hampshire Research Ethics Committee (REC reference: 280/99).All informed consent for the use of human material was provided in accordance with the Declaration of Helsinki.Mice were used in these studies as the least sentient species with an immune system comparable to humans.Following approval by local ethical committees, reporting to the Home Office Animal Welfare Ethical Review Board (AWERB) at the University of Southampton, in vivo experiments were conducted under UK Home Office Project licenses P81E129B7 and P4D9C89EA. A.R. receives funding from BioInvent International.Research by R.I.C is supported by use of equipment to measure body composition provided by SECA through a model industry collaborative agreement (mICA) with University Hospital Southampton.M.J.G previously acted as a consultant to a number of biotech companies and receives institutional payments and royalties from antibody patents and licenses.J.C.S has received funding from Roche.S.A.B acts as a consultant for a number of biotech companies and has received institutional support for grants and patents from BioInvent.M.S.C. acts as a consultant for a number of biotech companies, being retained as a consultant for BioInvent International and has received research funding from BioInvent, GSK, UCB, iTeos, and Roche. • fast, convenient online submission • thorough peer review by experienced researchers in your field • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Ready to submit your research Ready to submit your research ?Choose BMC and benefit from: ? Choose BMC and benefit from: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Supplementary Information", "section_num": null }, { "section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s13046-022-02294-5. ", "section_name": "Supplementary Information", "section_num": null }, { "section_content": "Authors' contributions K.H. designed and performed the experiments, analyzed and interpreted data and wrote the manuscript.R.L., R.C.G.S., K.T.J.M., M.G., S.M., K.L.S.C., R.J.O., R.B.F., S.J., S.G.B., T.M., L.N.D., C.E.H., R.S.K., J.L., J.D., H.M., S.J.C., R.J.S., M.J.C., C.H.O., R.I.C., R.R.F. and S.M.T. generated and provided key reagents, performed experiments, wrote specific method sections and analyzed data.A.R., B.F. and M.J.G. discussed and interpreted data and edited the manuscript, J.C.S., S.M.T., S.A.B. and M.S.C. acquired funding, designed the study, supervised data collection, discussed and interpreted data and edited the manuscript.The author(s) read and approved the final manuscript. ", "section_name": "Additional file 1:", "section_num": null }, { "section_content": "", "section_name": "Funding", "section_num": null }, { "section_content": "Anonymized leukocyte cones were sourced from healthy adult donors attending blood donation clinics at the National Blood Service (Southampton, UK).The use of leukocyte cones for this work was approved by the University of Southampton Faculty of Medicine Ethics Committee and the East of Scotland Research Ethics Service, Tayside, UK, Research ethical committee (REC) reference number: 16/ES/0048.Clinical samples from 6 anonymized mesothelioma patients (REC reference number: 13/SW/0128) and Donor matched Renal cell carcinoma (RCC) and non-cancerous healthy kidney tissue samples were obtained from resected kidneys from 5 RCC patients (REC reference number: 17/WA/0241).Lymphocele samples were sourced from 3 anonymized breast cancer patents (REC reference number: 10/H0504/73, for breast cancer patient samples).Peripheral blood samples were taken from Chronic Lymphoblastic Leukemia (CLL) patients and anonymized before experimental use (REC reference number: 10/H0504/187).These aforementioned clinical samples were released from the Human Tissue Authority Licensed University of Southampton, Cancer Sciences Tissue Bank, as approved by the Southampton and South West Hampshire Research Ethics Committee (REC reference: 280/99).All informed consent for the use of human material was provided in accordance with the Declaration of Helsinki.Mice were used in these studies as the least sentient species with an immune system comparable to humans.Following approval by local ethical committees, reporting to the Home Office Animal Welfare Ethical Review Board (AWERB) at the University of Southampton, in vivo experiments were conducted under UK Home Office Project licenses P81E129B7 and P4D9C89EA. ", "section_name": "Declarations Ethics approval and consent to participate", "section_num": null }, { "section_content": "A.R. receives funding from BioInvent International.Research by R.I.C is supported by use of equipment to measure body composition provided by SECA through a model industry collaborative agreement (mICA) with University Hospital Southampton.M.J.G previously acted as a consultant to a number of biotech companies and receives institutional payments and royalties from antibody patents and licenses.J.C.S has received funding from Roche.S.A.B acts as a consultant for a number of biotech companies and has received institutional support for grants and patents from BioInvent.M.S.C. acts as a consultant for a number of biotech companies, being retained as a consultant for BioInvent International and has received research funding from BioInvent, GSK, UCB, iTeos, and Roche. • fast, convenient online submission • thorough peer review by experienced researchers in your field ", "section_name": "Competing interests", "section_num": null }, { "section_content": "• support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year ", "section_name": "• rapid publication on acceptance", "section_num": null }, { "section_content": "At BMC, research is always in progress. ", "section_name": "•", "section_num": null }, { "section_content": "Ready to submit your research Ready to submit your research ?Choose BMC and benefit from: ? Choose BMC and benefit from: ", "section_name": "Learn more biomedcentral.com/submissions", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.1186/bcr920	Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells	Notch signaling has been implicated in the regulation of cell-fate decisions such as self-renewal of adult stem cells and differentiation of progenitor cells along a particular lineage. Moreover, depending on the cellular and developmental context, the Notch pathway acts as a regulator of cell survival and cell proliferation. Abnormal expression of Notch receptors has been found in different types of epithelial metaplastic lesions and neoplastic lesions, suggesting that Notch may act as a proto-oncogene. The vertebrate Notch1 and Notch4 homologs are involved in normal development of the mammary gland, and mutated forms of these genes are associated with development of mouse mammary tumors.In order to determine the role of Notch signaling in mammary cell-fate determination, we have utilized a newly described in vitro system in which mammary stem/progenitor cells can be cultured in suspension as nonadherent 'mammospheres'. Notch signaling was activated using exogenous ligands, or was inhibited using previously characterized Notch signaling antagonists.Utilizing this system, we demonstrate that Notch signaling can act on mammary stem cells to promote self-renewal and on early progenitor cells to promote their proliferation, as demonstrated by a 10-fold increase in secondary mammosphere formation upon addition of a Notch-activating DSL peptide. In addition to acting on stem cells, Notch signaling is also able to act on multipotent progenitor cells, facilitating myoepithelial lineage-specific commitment and proliferation. Stimulation of this pathway also promotes branching morphogenesis in three-dimensional Matrigel cultures. These effects are completely inhibited by a Notch4 blocking antibody or a gamma secretase inhibitor that blocks Notch processing. In contrast to the effects of Notch signaling on mammary stem/progenitor cells, modulation of this pathway has no discernable effect on fully committed, differentiated, mammary epithelial cells.These studies suggest that Notch signaling plays a critical role in normal human mammary development by acting on both stem cells and progenitor cells, affecting self-renewal and lineage-specific differentiation. Based on these findings we propose that abnormal Notch signaling may contribute to mammary carcinogenesis by deregulating the self-renewal of normal mammary stem cells.	[ { "section_content": "Stem cells in adult tissues are characterized by their ability to undergo self-renewal and multilineage differentiation [1].The elucidation of pathways that govern stem cell functions is essential for understanding normal development and organogenesis.Moreover, there is increasing evidence that defects in these pathways play an important role in carcinogenesis [2].The isolation of stem cells from the mammary gland in humans and rodents has been hindered by the lack of identified specific cell surface markers.Furthermore, investigation of the mechanisms underlying cell-fate decisions in mammary stem/progenitor cells has been limited by the lack of suitable in vitro culture systems, which maintain these cells in an undifferentiated state [3]. We have recently described an in vitro culture system that allows for the propagation of primary human mammary epithelial stem cells and progenitor cells in an undifferentiated ADAM = a disintegrin and metalloproteases; FACS = fluoresence-activated cell sorting; FBS = fetal bovine serum; FITC = fluorescein isothiocyanate; GSI = gamma secretase inhibitor; HBSS = Hanks Balanced Salt Solution; RT-PCR = reverse transcriptase-polymerase chain reaction. state, based on their ability to proliferate in suspension as spherical structures, which we have termed 'nonadherent mammospheres' [4].As has previously been described for neuronal stem cells and progenitor cells cultured as neurospheres [5], we have demonstrated that mammospheres are composed of stem cells and progenitor cells capable of self-renewal and multilineage differentiation [4].We have utilized this system to investigate the role of Notch signaling in mammary cell-fate determination. In other systems, Notch signaling has been shown to play an important role in cell-fate determination, as well as in cell survival and proliferation [6,7].The Notch proteins, represented by four homologs in mammals (Notch1-Notch4), interact with a number of surface-bound or secreted ligands (Delta-like 1, Delta-like 3, Delta-like 4, Jagged 1 and Jagged 2) [8][9][10].These interactions are modulated by modifier proteins from the Fringe family (Lunatic, Manic, and Radical Fringe) [11].Upon ligand binding, Notch receptors are activated by serial cleavage events involving members of the ADAM protease family, as well as an intramembranous cleavage regulated by gamma secretase (presinilin).This intramembranous cleavage is followed by translocation of the intracellular domain on Notch to the nucleus, where it acts on downstream targets [11].The vertebrate Notch4 gene has been shown to be involved in normal mammary development [12].In vitro, overexpression of a constitutively active form of Notch4 inhibits differentiation of normal breast epithelial cells [13].In vivo, transgenic mice expressing a constitutively active form of Notch4 fail to develop normal mammary glands and subsequently develop mammary tumors [14].These studies suggest the involvement of Notch signaling in normal breast development, and that alterations in Notch signaling may play a role in breast cancer development [15,16]. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Materials and methods", "section_num": null }, { "section_content": "Normal breast tissue from reduction mammoplasties was dissociated mechanically and enzymatically, as previously described [17].Cells were sieved sequentially through a 100 µm cell strainer and a 40 µm cell strainer (BD Falcon, Palo Alto, CA, USA) to obtain a single cell suspension. ", "section_name": "Dissociation of mammary tissue", "section_num": null }, { "section_content": "Single cells were plated on ultralow attachment plates (Corning, Acton, MA, USA) at a density of 20,000 viable cells/ml in primary culture and a density of 1000 cells/ml in passages.Cells were grown in serum-free mammary epithelial growth medium (BioWhittaker, Rockland, ME, USA), supplemented with B27 (Invitrogen, Carlsbad, CA, USA) and 10 ng/ml epidermal growth factor (BD Biosciences, Palo Alto, CA, USA).The bovine pituitary extract was excluded. Mammospheres were collected by gentle centrifugation (800 rpm) after 7-10 days, and were dissociated enzymatically (10 min in 0.05% trypsin, 0.53 mM EDTA-4Na; Invitrogen) and mechanically, using a fire-polished Pasteur pipette.The cells obtained from dissociation were sieved through a 40 µm sieve and analyzed microscopically for single cellularity.Cells plated at low densities (1000 cells/ml) were grown in conditioned medium from high-density primary cultures in suspension. ", "section_name": "Mammosphere culture", "section_num": null }, { "section_content": "Single cell suspensions were plated on collagen-coated plates at a density of 2000 viable cells/10 cm diameter dish.Cells were grown in Ham's F-12 medium (BioWhittaker) with 5% fetal bovine serum (FBS), 5 µg/ml insulin, 1 µg/ml hydrocortisone, 10 µg/ml cholera toxin (Sigma, St Louis, MO, USA), 10 ng/ml epidermal growth factor (BD Biosciences) and 1 × Pen/Strep/Fungizone Mix (BioWhittaker).Three-dimensional cultures in Matrigel were established as previously described [18]. ", "section_name": "Differentiating culture conditions", "section_num": null }, { "section_content": "To assess the lineage composition of the colonies, cells were fixed on plates for 20 min in methanol, at -20°C, and were then stained using Peroxidase Histostain-Plus and Alkaline-phosphatase Histostain-Plus kits (Zymed, South San Francisco, CA, USA), according to the manufacturer's protocol.The ductal-alveolar structures were fixed in Matrigel in 4% neutral formalin, and treated with 1 mg/ml proteinase K for 10-15 min at room temperature, prior to staining.For immunostaining in Matrigel, all antibody incubations were for 60-90 min at 37°C, and washes were for 20 min at 37°C, with a final overnight wash at 4°C.The primary antibodies -epithelial-specific antigen and cytokeratin 18 for epithelial cells, CD10 and cytokeratin 14 (Novocastra, Newcastle-upon-Tyne, UK) for myoepithelial cells -were used at the dilutions indicated by the manufacturer.The Notch4 antibody was used at a dilution of 1:50 for immunohistochemistry and of 1:20 for immunostaining structures within Matrigel.AEC and DAB (Zymed) were used as substrates for peroxidase, and NBT/BCIP (Gibco/ BRL, Gaithersburg, MD, USA) was used for alkaline phosphatase.FITC-labeled and Texas-red labeled secondary antibodies (Jackson Labs, West Grove, PA, USA) were used for fluorescent microscopy. ", "section_name": "Immunostaining", "section_num": null }, { "section_content": "MCF-7 cells were grown in RPMI 10% FBS on tissue culture-treated plastic dishes.Cells were collected by trypsinization and RNA was isolated as previously described and used to test expression of Notch1 and Notch4 by RT-PCR.MCF-7 cells expressed both Notch1 and Notch4 so they were further used for the gene reporter assay.Expression of Notch4 in MCF-7 cells was also confirmed by immunohistochemistry.The staining was mostly nuclear and cytoplasmic, although membrane staining was also present, confirming detectable levels of active Notch4 in MCF-7 cells. The specificity of the antibody was tested by the manufacturer via western blotting using cell lysates from a variety of human cancer cell lines (Jurkat cells, HeLa, H4, etc.) that express all four homologs of Notch.The antibody was raised against a synthetic peptide that does not show sequence similarity with any of the other Notch members, as shown by Blast search analysis.Using the Lipofectamine-Plus kit (Promega, Madison, WI, USA), cells were co-transfected with pcDNA3 (Invitrogen) and a pGL3-promoter vector (Promega) containing a Hes-1 promoter-derived sequence upstream of luciferase, according to the manufacturer's protocol.Stable transfection was achieved by selecting with 800 µg/ml Geneticin (Gibco/ BRL). Cells were incubated for 4, 8 and 16 hours in the presence of agonists or antagonists of Notch signaling as follows: blocking Notch4 antibody (Santa-Cruz, Santa Cruz, CA, USA) was used in dilutions of 1:100, 1:50, 1:25 and 1:10, gamma secretase inhibitor (GSI) type I (Z-Leu-Leu-Nle-CHO; Calbiochem, San Diego, CA, USA) was used at 20 µM, 10 µM, 5 µM, 1 µM, 100 nM and 10 nM concentration, and the DSL peptide (CDDYYYGFGCNKFCRPR; Research Genetics, Huntsville, AL, USA) was used at 10 µM, 1 µM, 100 nM, 10 nM and 1 nM.The control scrambled peptide (DYGKRCYGCFPDYFNCR) was used at 10 µM concentration.Dimethylsulfoxide 1:50 was used as a control for GSI.Direct evidence that GSI blocks Notch4 and Notch1 cleavage in vitro has been provided previously [19]. The treatment with antagonist involved incubating for 1 hour at 37°C, prior to treatment with the DSL peptide.Incubation of the antibody with the peptide against which it was raised (Santa Cruz) was carried out at antibody to peptide ratios of 1:1, 1:2.1:3 and 1:4 (vol:vol).Luciferase activity was assessed using the Luciferase assay system (Promega) and a Turner Designs luminometer TD-20/20 (Turner Designs, Sunnyvale, CA, USA). ", "section_name": "Luciferase gene reporter assay", "section_num": null }, { "section_content": "Treatment with Notch agonists and antagonists was carried out as described for the reporter assay.The DSL peptide was used at a concentration of 100 nM.The recombinant Delta 1 Fc ligand was prepared and used as previously described [20].Anti-human Fc antibody (Jackson Labs) was added to the medium at 1:100 dilution, to cluster the recombinant ligand-receptor complexes.The blocking Notch4 antibody was used at a dilution of 1:50, and the GSI at a concentration of 5 µM.In experiments in which treatment was applied for longer than 48 hours, it was added every 2 or 3 days when the medium was changed.The Notch4 antibody and antigenic peptide used for treating cells in culture did not contain sodium azide. ", "section_name": "Treatment with Notch agonists and antagonists", "section_num": null }, { "section_content": "Cells were fixed in methanol and immunostained with antiepithelial-specific antigen, CD10 and Muc1 antibodies (Novocastra), at the concentration recommended by the manufacturer.Incubation was performed for 30 min at room temperature in Hanks Balanced Salt Solution (HBSS) with 2% FBS, followed by washing in HBSS with 2% FBS.The same procedure was followed for staining with secondary antibody, anti-goat IgG or anti-rabbit IgG, labeled with FITC (1:200 dilution; Jackson Labs).After incubation, cells were washed once with HBSS (BioWhittaker) and were resuspended in HBSS supplemented with 5% FBS.Cells were stained with 1 µg/ml propidium iodide (Sigma) for 30 min for DNA cell cycle analysis or for 5 min for viability.Analysis was performed using FACStar PLUS (Becton Dickinson, Palo Alto, CA, USA) and the Elite SP (Becton Dickinson) flow cytometer. ", "section_name": "FACS analysis", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "In order to investigate the role of the Notch pathway in the development of the mammary gland, we examined the effect of agonists and antagonists of Notch signaling on cell-fate determination utilizing mammary stem/progenitor cells cultured in suspension, as mammospheres, and differentiated mammary cells, obtained by passaging these cells on a collagen substratum.The experimental strategy is outlined in Fig. 1.The activators and inhibitors of Notch signaling were applied at three different stages of in vitro development, which we have previously characterized [4].First, the treatment was applied in suspension culture, which is enriched in stem/progenitor cells (self-renewal of stem cells and the proliferation of progenitor cells occur in these conditions) (Fig. 1a).Second, the treatment was applied in cultures on collagen substratum, under conditions that promote differentiation of mammosphere-derived cells (multipotent progenitors commit to a specific lineage) (Fig. 1b).Finally, the treatment was applied in cultures on a collagen substratum, using differentiated cells passaged on a collagen substratum (lineage-restricted progenitors undergo terminal differentiation) (Fig. 1c). A synthetic peptide derived from the DSL (Delta-Serrate-LAG 2) domain conserved in all Notch ligands [9] and a previously described recombinant Delta 1 ligand fused to the immunoglobulin Fc fragment (used in combination with an anti-human Fc antibody, for ligand clustering) [20] were utilized as agonists of Notch signaling.A Notch4-specific antibody or a GSI that blocks the intramembranous cleavage of Notch, required for signaling [21], was used as the antagonist.The synthetic DSL peptide, the recombinant Delta fused to Fc and GSI are all well characterized with respect to their effect on Notch signaling [9,20].We utilized a reporter assay in MCF-7 cells, in which luciferase expression was driven by the Hes-1 promotor, a gene regulated by Notch signaling [22], to confirm the ability of activating or inhibiting ligands to affect Notch signaling. As shown in Fig. 2a, DSL peptide upregulated Hes-1driven luciferase expression.These results are consistent with previous characterization of these Notch signaling agonists in reporter assays and in experiments using other cell types [9].Notch signaling was blocked using a Notch4-specific antibody, or a GSI, which abrogates the intramembranous cleavage of Notch, required for signaling [19,21].Both of these inhibitors blocked endogenous Hes-1-driven luciferase expression, as well as the increase in luciferase expression induced by the DSL ligand (Fig. 2a).The specificity of the blocking antibody was confirmed by successful competition with the antigenic peptide used to produce the antibody (data not shown).Furthermore, these effects were dose dependent (Fig. 2b). ", "section_name": "Experimental strategy", "section_num": null }, { "section_content": "We examined the effects of these agonists and antagonists of Notch signaling on primary, secondary and tertiary mammosphere formation (Fig. 1a).This assay has previously been used for the in vitro study of neural stem cell selfrenewal [19] and relies on the observation that neurosphere formation is initiated clonally by neural stem cells [5]. Utilizing retroviral marking studies, we also have demonstrated that mammospheres are clonally derived and do not result from cellular aggregation [4].Primary, secondary, and tertiary generation mammospheres were formed in the presence or absence of the Notch-activating DSL peptide.Although DSL peptide had only modest effects on primary mammosphere formation, it increased secondary and tertiary mammosphere formation 10-fold, compared with control cultures (Fig. 3a,3b,3d,3e,3g).If this effect was due to stem cell self-renewal, we would predict that cells derived from these spheres would retain their multipotent differentiation ability.We therefore examined the ability of secondary and tertiary mammosphere-derived cells to clonally differentiate along the multiple lineages present in the adult mammary gland. The adult mammary gland has a lobuloalveolar structure composed of three cell lineages: myoepithelial cells that form the basal layer of ducts and alveoli, ductal epithelial cells that line the lumen of ducts, and alveolar epithelial cells that synthesize milk proteins [23].Mammospheres grown in the presence or absence of Notch agonists or antagonists were dissociated into single cells and plated at clonogenic densities on collagen substrata in a medium that promotes differentiation [4].After 7 days of cultivation, the clonally derived colonies were immunostained using lineage-specific markers (epithelial-specific antigen and cytokeratin 18 for ductal epithelial cells, and cytokeratin 14 and smooth muscle actin for myoepithelial cells).The number of multipotent cells after one passage was seven times higher, and that after two passages 100 times higher, in mammospheres cultured in the presence of DSL, Effect of modulators of Notch signaling on self-renewal of mammary stem cells and on lineage commitment of mammary progenitor cells: experimen-tal strategy Effect of modulators of Notch signaling on self-renewal of mammary stem cells and on lineage commitment of mammary progenitor cells: experimental strategy.Treatment was applied (a) to mammospheres in suspension culture, (b) to mammosphere-derived cells cultivated on a collagen substratum, and (c) to differentiated cells cultivated on a collagen substratum.The assay for self-renewal was mammosphere formation.The assay for multilineage potential or lineage commitment was lineage-specific immunostaining of clonogenic cultures on the collagen substratum.compared with mammospheres cultured without added DSL.These calculations are based on the number of spheres (Fig. 3g) and the corresponding percentage of multilineage colonies, in each experiment (Fig. 3h). The ability of Notch activating ligands to increase mammosphere formation, as well as the production of multilineage progenitors, suggests that Notch activation promotes mammary stem cell self-renewal.Similar effects to the DSL peptide on mammosphere formation were obtained with recombinant Delta Fc fragment (data not shown).In addition to increasing the number of mammosphere initiating cells and bipotent progenitor cells, treatment with Notch agonists increased their proliferation potential, as shown by the increased size of mixed lineage colonies, generated clonally on collagen culture by these cells (Fig. 3i).Treatment with Notch agonists was applied in suspension culture only, and was not applied to sphere-derived cells subsequently cultured on collagen substrata, suggesting that the effect on proliferation is irreversible.This positive effect of Notch activation on proliferation potential of stem/ progenitor cells also occurs in the mammospheres, as demonstrated by an increased size of the primary and secondary mammosphere diameter in the DSL-treated suspension cultures (mean ± standard error of the mean, 245 ± 11.5 µM versus 130 ± 3 µM; n = 69 and n = 89).This is due to an increased number of cells per sphere, as demonstrated by cell counts that are approximately double in DSL-treated mammospheres compared with control cul-tures.The viability of cells within a mammosphere was similar in the presence of and the absence of DSL.These results indicate that the Notch activation increases proliferation of progenitor cells. In order to further investigate the role of Notch signaling, and in particular Notch4, in mammary stem cell selfrenewal, we assayed the effect of Notch inhibitors on mammosphere formation.The addition of a Notch4 blocking antibody to primary cultures had no effect on primary sphere formation, but completely abolished secondary mammosphere formation (Fig. 3a,3c,3d,3f,3h).A similar effect was obtained by blocking Notch activation with a GSI at 1-5 µM, a concentration found to effectively block Notch activation in the luciferase reporter assay (Fig. 2a).In contrast to the effect of adding Notch4 blocking antibody at the time of mammosphere formation, this treatment had no effect when added 24-48 hours after mammosphere formation.This suggests that the effects of Notch activation on mammary stem cell self-renewal may occur during the initial stages of mammosphere formation.The cells responding to modulation of Notch signaling are the sphere initiating cells, capable of survival and proliferation in suspension. ", "section_name": "Effects of Notch signaling on self-renewal of mammary stem cells", "section_num": null }, { "section_content": "In other developing systems, Notch signaling has been shown to effect lineage commitment as well as stem cell self-renewal [20,[24][25][26].The inability of previously described culture systems of mammary cells to maintain progenitor cells in an uncommitted state has limited the in vitro study of lineage commitment.Since we have previously demonstrated that, in addition to mammary stem cells, mammospheres contain mammary progenitors capa-ble of multilineage differentiation, we utilized this system to determine whether Notch signaling could also act on mammary progenitor cells to affect lineage-specific commitment.In order to test this, we added the DSL peptide to mammosphere cultures (Fig. 1a), to cultures of mammosphere-derived cells on collagen substrata in conditions that Effect of modulators of Notch signaling on self-renewal of mammary stem cells and on lineage commitment of mammary progenitor cells Effect of modulators of Notch signaling on self-renewal of mammary stem cells and on lineage commitment of mammary progenitor cells.(a)-(g) Effect of Notch agonist and antagonist treatment on primary and secondary mammosphere formation.Primary and secondary mammospheres were grown in standard conditions (a, d), in the presence of DSL peptide (b, e) and in the presence of N4 blocking antibody (N4Ab) (c, f).Primary mammospheres grown in the presence of N4 antibody (c) are smaller than the control (a).Secondary mammosphere formation is completely blocked by treatment with Notch4 blocking antibody (d, f).The addition of DSL peptide increases primary, secondary and tertiary mammosphere formation (a, b, d, e, g).Data are presented as the mean ± standard error of the mean.The calculated number of multilineage progenitor cells (number of spheres × % multilineage progenitors) shows a sevenfold increase after one passage, and a 100-fold increase after two passages, in mammospheres cultured in the presence of DSL compared with mammospheres cultured without added DSL. (h)-(k) Effect of Notch activation on lineage specification of human mammary progenitor cells.DSL treatment of mammospheres in suspension culture (DSL1) and on mammosphere-derived cells on the collagen substratum (DSL2) increases the number of myoepithelial progenitors, as shown by the clonogenic assay (h), and increases the rate of proliferation of bipotent and myoepithelial progenitors, as reflected by the colony size (i).DSL treatment in suspension culture increases the percentage of myoepithelial cells, as shown by flow-cytometry analysis (j).Cells were stained red (phycoerythrin [PE]) with myoepithelial marker CD10, and green (FITC) with the ductal epithelial marker epithelial-specific antigen (ESA).Treatment with DSL peptide does not have a lineage selective effect on differentiated cells, as shown by flow-cytometry analysis of human mammary epithelial cells passaged twice on collagen (k).Data are presented as the mean ± standard error of the mean.promote their differentiation (Fig. 1b) or to cultures of differentiated cells (Fig. 1c). Cells were assayed for lineage-specific commitment, using the clonogenic assay described earlier.As shown in Fig. 3h (DSL1), the percentage of myoepithelial progenitor cells generated by DSL-treated mammospheres was increased approximately 13-fold compared with cells from mammospheres cultured in the absence of DSL ligand.The percentage of myoepithelial progenitor cells generated by mammosphere-derived cells treated with DSL starting from the moment of their plating on collagen culture (DSL2) generated sevenfold more myoepithelial progenitor cells compared with controls.The significant effect of DSL peptide, when added only to suspension-cultured mammospheres, suggests that Notch activation increased the number of myoepithelial progenitors and/or their rate of proliferation in an irreversible manner.The increased size of myoepithelial and bilineage colonies (Fig. 3i) formed in the presence of DSL peptide suggests that Notch stimulation may promote proliferation of myoepithelial and bilineage progenitors. In order to determine whether the increase in colony size results from an effect of Notch on cell survival, we used propidium iodide staining in the same clonogenic assay.Treatment with DSL peptide did not have a discernable effect on the cell-death rate of either ductal epithelial cells or myoepithelial cells.To assess more accurately the increase in total number of myoepithelial cells upon treatment with DSL in suspension culture, we repeated the same experiment described earlier, utilizing flow cytometry analysis, to quantitate the cell lineage (Fig. 3j).The results showed a 15-fold increase in myoepithelial cells upon addition of DSL peptide to the culture. ", "section_name": "Effect of Notch signaling on lineage specification of mammary progenitor cells", "section_num": null }, { "section_content": "In contrast to the significant effects of Notch signaling on uncommitted mammary cells, modulation of the Notch pathway had no significant effect on differentiated mammary epithelial cells.The addition of DSL peptide to differentiated cells cultured on collagen substrata (Fig. 1c) had no effect on lineage specification, as shown by flow-cytometry analysis using lineage-specific markers (Fig. 3k).Furthermore, the addition of Notch antibody or the GSI at 1-10 µM had no significant effect on cell number or lineage commitment (data not shown).This also demonstrates that Notch activation is not necessary for the survival of fully differentiated mammary cells. ", "section_name": "Effect of Notch signaling on differentiated mammary epithelial cells", "section_num": null }, { "section_content": "It has previously been demonstrated that culture of human or rodent mammary cells in reconstituted basement membrane (Matrigel) promotes morphogenic differentiation [18,27].Since mammospheres are composed of undiffer-entiated mammary cells, we have utilized three-dimensional Matrigel cultures to explore the morphogenic differentiation potential of these cells.Following 3-4 weeks of cultivation in Matrigel, mammospheres develop extensive ductal lobuloalveolar structures similar in morphology to those found in vivo (Fig. 4a,4b).We utilized this system to examine the role of Notch signaling in morphogenesis.Secondary mammospheres were imbedded in Matrigel and treated with DSL peptide, Notch4 antibody or GSI.Treatment with DSL peptide promoted earlier development, as well as increased length and number of branching structures, compared with control cultures (Fig. 4c,4d).In contrast, the Notch4 antibody completely inhibited branching morphogenesis (Fig. 4c,4d).Similar effects were produced by blocking Notch signaling with GSI (data not shown). ", "section_name": "Effect of Notch signaling on branching morphogenesis", "section_num": null }, { "section_content": "We and other workers have found that expression of Notch receptors in the adult human mammary gland was below the level of detection using immunostaining of tissue sections, but was detectable by RT-PCR [28].Moreover, expression of Notch4 in dissociated mammary tissue was below the level of detection using flow cytometry.The experiments already described, however, suggested that Notch receptors are present in mammary stem cells and progenitor cells, and are downregulated in differentiated cells.Previous gene expression profile analysis by microarray and real-time RT-PCR showed that mammospherederived cells passaged in suspension culture express all Notch receptors at a higher level (twofold to fourfold) than the same cells cultured under conditions that induce differentiation [4]. These results suggest that Notch receptors are expressed at higher levels in more primitive mammary cells, such as those found in the developing mammary gland, compared with the mature gland.To test this hypothesis, we immunostained structures generated by mammospheres in Matrigel culture, at different stages of growth (between 4 days and 2 weeks).We found that cells expressing Notch4 were uniformly distributed in spherical structures at the beginning of cultivation (Fig. 5a,5b).As branching morphogenesis occurred, Notch4 expression became limited to the subterminal end of branches (Fig. 5c,5d).The corresponding areas in vivo have previously been postulated to contain mammary stem cells and progenitor cells [23]. ", "section_name": "Expression of Notch4 in human mammary epithelial cells in vitro", "section_num": null }, { "section_content": "In order to study the role of Notch signaling in mammary cell-fate determination, we have utilized an experimental system that allows the analysis of cell self-renewal and differentiation in vitro.This enabled us to study the effect of Notch activation and inhibition at a single cell level and under conditions where the timing and the duration of Notch signaling could be controlled.Our findings suggest that Notch signaling regulates cell-fate decisions in the mammary gland at several distinct developmental stages. We propose that in the mammary gland, as has been reported in other developmental systems, Notch activation acts as a regulator of asymmetric cell-fate decisions.Notch activation promotes self-renewal in stem cells, whereas in later stages of development it biases cell-fate decisions in mammary progenitor cells towards adoption of a myoepithelial cell fate versus an epithelial cell fate. The increase in the number of mammosphere-initiating cells reflects an increase in the number of cells capable of selfrenewal, in the experimental system used in this study.The effect of Notch activation on mammosphere formation underscores the value of the mammosphere cultivation system in studying in vitro self-renewal regulation.It has previously been shown in suspension culture that neural stem cells can generate mixed colonies, termed neurospheres, composed of stem cells and progenitor cells.We have demonstrated that mammospheres are composed of stem cells and progenitor cells capable of self-renewal and multilineage differentiation [4].Our clonal analysis of individual cells from single spheres suggested that mammospheres are composed of approximately 150-200 progenitor cells, which cannot generate new spheres but have bilineage or trilineage differentiation potential, and of one or two putative stem cells able to generate a new sphere with the same cellular composition as the parental sphere.The constant frequency of sphere formation during multiple passages, observed in passages of both individual and pooled mammospheres, suggests that only asymmetric self-divisions or a small number of symmetric self-renewal divisions contribute to sphere formation (Fig. 6a).If bipotent or monopotent progenitor cells were able to survive in suspension, they would generate mammospheres of increasingly smaller size and, eventually, they would stop growing in suspension after several passages (Fig. 6b), which did not occur in the experimental setting used here.An increase in the number of symmetric self-renewal divisions would result in an amplification of the cellular population, by progressively increasing the number of mammospheres at each passage (Fig. 6c).This effect was obtained upon exogenous activation of Notch signaling.In this discussion we adopted the operational definition of stem cells as the only normal cells capable of self-renewal [29,30]. Treatment with Notch agonists increased the commitment of bilineage progenitors to myoepithelial lineage, when applied early in suspension culture (Fig. 1, DSL1) or later on collagen culture (Fig. 1,DSL2).The effect on lineage commitment appears to be irreversible, since early activation of Notch, in suspension culture, resulted in an increase in the number of myoepithelial progenitors -although the treatment with Notch agonists was not maintained on collagen cultures.In addition to an increase in the number of bipotent and myoepithelial progenitor cells, treatment with Notch agonists resulted in a significant increase in the size of both mixed and myoepithelial colonies.This sug-gests that Notch activation increases the proliferation potential of both bipotent and myoepithelial progenitors.These effects are irreversible and may involve an instructive mechanism or the selective proliferation of myoepithelial cells.In contrast to the effects of Notch signaling on mammary stem cells and progenitor cells, activation or inhibition of these pathways in fully differentiated mammary cells had no apparent effect. The increased branching we observed in the three-dimensional cultivation system upon Notch activation is consistent with previous studies demonstrating that proliferation of myoepithelial cells can facilitate branching morphogenesis.Moreover, Notch signaling appears to be absolutely required for branching and growth in three-dimensional cultures as development of branching structures is completely blocked by Notch4 antibody or GSI.The expression of Notch4 protein in the growing mammary tree in vitro is also consistent with an important role of Notch signaling in morphogenesis of the mammary gland.The apparent discrepancy between our results and the previous findings by Uyttendaele and colleagues [13] (who showed that overexpressing Notch4 in the mouse mammary epithelial TAC-2 cell line prevented branching morphogenesis in response to hepatocyte growth factor or transforming growth factor beta) underscores the importance of the experimental systems utilized.This discrepancy may be accounted for by differences in the experimental systems, such as the use of an immortalized mouse epithelial cell line in the Uyttendaele study compared with primary normal human cells in our study.Furthermore, Uyttendaele and colleagues utilized overexpression of activated Notch, whereas we used exogenous modulation of the Notch pathway. Smith and colleagues [12] demonstrated that Notch4 has an important role in both normal mammary development and carcinogenesis.Transgenic mice harboring a constitutively active Notch4 under mouse mammary tumor virus promoter regulation exhibited arrested mammary gland development, and eventually developed poorly differentiated adenocarcinomas.Questions remain in interpreting the report that the Notch4 knockout mouse has no apparent defect in mammary gland development [31].This may indicate that in vivo there are compensatory mechanisms absent in our in vitro experimental system.Alternatively, the expression or activity of different homologs of Notch can be interdependent, as suggested by previous studies [32]. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Our findings regarding the role of Notch in promoting selfrenewal of mammary stem cells, in addition to previous observations that it can function as a proto-oncogene [15,16,28,[33][34][35][36][37][38], suggest that abnormal Notch signaling may be involved in carcinogenesis, through deregulation of normal mammary stem cell self-renewal.Indeed, it has recently been proposed that stem cells or early progenitor cells constitute the primary targets of transformation [2]. We have recently described the existence of a tumorigenic subset of human breast carcinoma cells, with a definable phenotype, which is responsible for tumor formation as well as for generating the phenotypic heterogeneity found in breast cancers [39].Tumorigenic cells, like their normal stem cell counterparts, can therefore undergo self-renewal as well as differentiation, albeit aberrant.We have postulated that the tumor stem cell population drives tumorigenesis, recurrence and metastasis [40]. In addition to our findings regarding the role of Notch in self-renewal of mammary stem cells, these findings suggest that carcinogenesis may involve the deregulation of processes involved in normal stem cell self-renewal.Elucidation of key elements of these pathways may identify new targets for cancer therapy [32]. ", "section_name": "Conclusions", "section_num": null } ]	[ { "section_content": "Thanks are due to Dr Thomas Giordano for tissue procurement, the University of Michigan Cancer Center Flow Cytometry core, Dr Michael Clarke for technical advice and Dr Sean Morrison for providing the recombinant Delta 1-Fc recombinant ligand and for critical review of the paper.This work was supported by NIH grants CA66233 and CA101860, and in part by the University of Michigan Cancer Center NIH Support Grant 5 P 30 CA46592. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "MW has financial holdings in Oncomed, which has applied for a patent on cancer stem cell technologies. ", "section_name": "Competing interests", "section_num": null } ]
10.17219/acem/128764	NF-κB1 -94del/del ATTG polymorphic variant maintains CLL at an early, mildest stage	NF-κB is an essential player in cancer biology, especially in tumor development, due to its constitutive activation, and because a four-base deletion (ATTG) in the NF-κB1 promoter region at site -94, alters mRNA stability and regulates translation efficiency. This polymorphism is a good candidate risk marker and modulator of clinical course in chronic lymphocytic leukemia (CLL). As the effect of this NF-κB1 gene polymorphism has not been studied in patients with CLL so far, the present study was undertaken to find out whether the NF-κB1 promoter -94 ins/del ATTG polymorphism might be an essential genetic risk factor and/or modulatory disease player associated with CLL.The NF-κB1 -94 ins/del ATTG (rs28362491) polymorphism was investigated as a potential CLL susceptibility and progression factor, along with demonstration of potential modulation of the stage of clinical disease based on Rai classification.The associations of NF-κB1 -94 ins/del ATTG polymorphism with CLL and its clinical manifestation in 282 Polish individuals, including 156 CLL patients, were analyzed using polymerase chain reaction (PCR) with primers including a labeled forward primer, followed by capillary electrophoresis.A higher occurrence of the del/del homozygosity was observed among patients when compared to controls, resulting in an increase in CLL risk of more than twofold in patients carrying this homozygous genotype (OR = 2.23, p = 0.02, 95% CI = 1.14-4.37). Moreover, the del/del-positive patients more frequently presented the less aggressive disease phenotype (Rai 0), suggesting a low probability of progression to more advanced disease.The NF-κB1 -94 del/del genetic variant, although associated with increased risk of CLL disease, may be associated with maintenance of disease severity in the early, mildest stage. The likelihood of disease progression may increase as the frequency of wild-type (insertion) alleles for this polymorphism increases.	[ { "section_content": "Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the western world, with an incidence rate of 4-5/100,000.Multifactorial pathogenesis creates a complicated interaction network of internal (genetic and epigenetic) and external (for instance, microenvironmental and antigenic stimuli) factors engaged in the transformation, progression and evolution of CLL. 1 Most CLL cells are inert and are arrested in G0/G1 of the cell cycle, due to the inhibition of apoptosis associated with the increased expression of anti-apoptotic proteins from the BCL2 and IAP families, and the reduced expression of BAX and BAK, pro-apoptotic proteins from the BCL2 family.However, it is the progressive accumulation of tumor cells that ultimately leads to symptomatic disease.Chronic lymphocytic leukemia has a heterogeneous outcome, in which some patients progress rapidly and have short survival prognoses, whereas others have a more stable clinical course that may not need treatment for years.3][4][5] These systems define the following stages: 1) early stage (Rai 0, Binet A); 2) intermediate stage (Rai I/II, Binet B); and 3) advanced stage (Rai III/IV, Binet C).However, these systems are of limited prognostic value and the biological mechanisms involved in the clinical progression from early stages of patients with chronic lymphocytic leukemia are not well known. Among the genetic factors related to CLL, the pleiotropic transcription factor nuclear factor-kappa B1 (NF-κB1; p105/p50), a potential key contributor to tumorigenesis in many types of cancer, plays an important role.This redox-sensitive transcription factor regulates a number of inflammatory genes, and is an essential player in the initiation and progression of pathogenesis of many autoimmune and inflammatory diseases [6][7][8] as well as a tumorigenesis promotor. 9It regulates the transcription of a wide range of genes involved in the immune response, cell adhesion, differentiation, proliferation, angiogenesis, cellular stress reactions, tumorigenesis, and cell survival and apoptosis. 10Specifically, the active form of the NF-κB1 gene product, protein p50, can act as a transcription factor to regulate a target gene. 11,12everal investigators have reported the constitutive activation of NF-κB in various tumor cells and cell lines, such as in breast cancer, 13 colorectal cancer, 14,15 lung cancer, 16,17 pancreatic cancer, 18,19 melanoma, 11 and multiple myeloma. 20Most importantly, the activation of the NF-κB pathway mediates CLL cell survival and is further emerging as a prognostic marker in CLL. 1,21F-κB1 (Gene ID: 4790) is mapped to chromosome 4 locus 4q23-q24 22 (Fig. 1) and encodes the non-DNA-binding cytoplasmic molecule p105 and a DNA-binding protein corresponding to the N-terminus of p105-p50 (OMIM: 164011).This gene is composed of 24 exons and introns varying between 40,000 and 323 bp, spanning 156 kb (Fig. 1).Interindividual genetic variation is considered to be an important cancer risk and/or modulatory factor, and may impact the expression pattern encoded by the gene.Its location, molecular structure and genetic susceptibility have been extensively studied.Several variations have been described within the master regulator at the center of the NF-κB1 gene, with special interest given to rs72696119 (C>G), rs28362491 (-94 ins/del ATTG), rs4648068 (A>G), and rs12509517 (G>C). ", "section_name": "Background", "section_num": null }, { "section_content": "A common insertion/deletion (-94 insertion/deletion ATTG, rs28362491), located between 2 putative key promoter regulatory elements (Fig. 1) (activator protein 1 and κB binding site), has a functional impact and is the most widely investigated. 20This modification occurs between 2 important regulatory elements in the promoter region of the NF-κB1 gene.The deletion of 4 bases (ATTG) reduces or prevents binding to nuclear proteins, and leads to lower transcript levels of the NF-κB1 gene, thereby changing mRNA stability and regulating translation efficiency. 20either numerous case-control studies [23][24][25][26][27] nor several meta-analyses [28][29][30][31][32][33][34] have resolved the controversy of a potential association between the NF-κB1 -94 ins/del ATTG promoter polymorphism and cancer risk. ", "section_name": "NF-κB1 -94ins/delATTG (rs28362491)", "section_num": null }, { "section_content": "We undertook this study to find out whether the -94 ins/ del ATTG polymorphism in the NF-κB1 promoter might be an essential genetic risk factor and/or modulatory disease player associated with chronic lymphocytic leukemia. ", "section_name": "Objectives", "section_num": null }, { "section_content": "", "section_name": "Materials and methods", "section_num": null }, { "section_content": "The NF-κB1 promoter -94 ins/del ATTG (rs28362491) polymorphism was determined in a group of 282 Polish individuals, including 156 patients with CLL diagnosed at the Department of Hematology, Wroclaw Medical University, Poland.Chronic lymphocytic leukemia was diagnosed according to defined clinical, morphological and immunological criteria.At the time of the study recruitment, all patients were untreated.Due to the availability of clinical data, the Rai classification was included for 144 patients.The clinical characteristics of the study group are shown in Table 1.One hundred and twenty-six age-and sex-matched healthy individuals that we previously genotyped 35 were included as a control group. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the Declaration of Helsinki (1975), as revised in 2008.The study was approved by the Wroclaw Medical University Ethics Committee.Informed consent was obtained from all individual participants included in the study. ", "section_name": "Patients and controls", "section_num": null }, { "section_content": "Genomic DNA from lymphocytes was isolated using a DNA extraction kit (NucleoSpin Blood; Macherey-Nagel GmbH & Co., KG, Düren, Germany) following the manufacturer's instructions. ", "section_name": "Genotyping DNA isolation", "section_num": null }, { "section_content": "The primer sequences for the DNA amplification reactions were designed based on information reported by Zhou et al. 11 and confirmed using the National Center for Biotechnology Information (NCBI) database (Table 2).The 5' end of the sense primer was labeled with TAMRA (Table 2). Polymerase chain reaction (PCR) was carried out in a total volume of 10 μL.The reaction mixture contained 30 μg of genomic DNA, 0.1 μM each of sense primer (forward) (Metabion International AG, Planegg, Germany) and antisense primer (reverse) (Genomed S.A., Warszawa, Poland), 1 U Taq polymerase (EURX, Gdańsk, Poland), 1× PCR buffer (containing 15 mM MgCl 2 ) (EURX), and buffered dideoxynucleotide mixture (dNTP) containing 200 μM of each dNTP (Invitrogen, Life Technologies/Thermo Fisher Scientific, Foster City, USA).Amplifications were performed in a T100 ™ Thermal Cycler (Bio-Rad, Hercules, USA).The PCR cycling conditions were as follows: 95°C for 300 s, followed by 34 cycles of 94°C for 18 s, 64°C for 18 s, 72°C for 18 s, and 72°C for 600 s.Qualitative analysis of PCR products was based on electrophoresis in 2% agarose gel, visualized under ultraviolet light (UV). Polymorphism analysis was performed using capillary electrophoresis using an Applied Biosystems ® 3130 Genetic Analyzer, equipped with 3130 Series Data Collection Software v. 4 (Life Technologies/Thermo Fisher Scientific). ", "section_name": "Identification of -94 ins/del ATTG polymorphism (rs28362491) in the NF-ĸB1 gene", "section_num": null }, { "section_content": "All genotypes were tested for deviations from Hardy-Weinberg equilibrium (HWE) using the χ 2 test.Allele and genotype association analyses were performed using dominant and recessive genetic models.Genotype and allele analyses were performed using SHEsis software (http:// analysis.bio-x.cn/myAnalysis.php). The odds ratio (OR) and 95% confidence intervals (95% CI) were calculated using the Simple Interactive Statistical Analysis platform (SISA, http://www.quantitativeskills. com/sisa/). Differences were considered as statistically significant if the p-value was <0.05. ", "section_name": "Statistical analyses", "section_num": null }, { "section_content": "Genotype distributions did not deviate from HWE, either in the patients or the controls in our cohort of Polish CLL patients (Table 3).A univariate analysis showed that patients with a recessive polymorphic homozygous NF-ĸB1 -94 del/del variant (NF-κB1 rs28362491) have a significantly modified risk of CLL.We observed that the del/ del genotype of this polymorphism increased the risk of the disease 2.23-fold (χ 2 = 5.63, p = 0.02, OR = 2.23, 95% CI = 1.14-4.37;Table 4). Additional associative analysis of the -94 ins/del ATTG polymorphism (rs28362491) in the NF-κB1 gene in CLL patients using Rai stage was carried out in a subset of 144 patients due to the limited availability of clinical data.The results are shown in Table 5. Patients with stages III and IV were analyzed together due to the small size of the groups.The CLL-prone recessive del/del genotype (χ 2 = 5.63, p = 0.02, OR = 2.23, 95% CI = 1.14-4.37)(Table 4) was the most frequent variant in CLL patients with Rai stage 0 (27.1% > 20% > 14.3% > 18.2%; Table 5). Statistical analysis of the genotype distribution showed differences in genotype frequencies between patients with the following stages: 0 compared to I (χ 2 = 7.65, p = 0.02); I compared to II (χ 2 = 6.78, p = 0.03); 0 compared to control group (χ 2 = 8.00, p = 0.02); and I compared to control group (χ 2 = 7.65, p = 0.02).The ins/ins genotype may protect against progression of the disease, favoring maintenance in the initial stage (Rai 0).The recessive genotype had significantly different frequencies in the healthy group and the group of patients with the lowest clinical severity (Rai 0) (χ 2 = 7.58, p = 0.006, OR = 2.98, 95% CI = 1.34-6.62);The incidence of this genotype was the highest in the group with Rai 0. ", "section_name": "Results", "section_num": null }, { "section_content": "NF-κB is an essential component of cancer biology, affecting proliferation, apoptosis, vascular regeneration, inflammation, and metastasis, as well as infiltration, especially in tumors with NF-κB constitutive activation, such as those with a hematopoietic origin.Regulation of over 200 different genes by NF-κB serves as the main signal for cell survival, participating in many stages of carcinogenesis and cancer cell resistance to chemo-and radiotherapy.This has therefore become an important target of associative research on cancer pathogenesis and autoimmune diseases. 11 relationship between NF-κB1 genetic polymorphism and cancer risk has been identified, involving polymorphism variants rs72696119 (C>G), rs28362491 (-94 ins/del ATTG), rs4648068 (A>G), and rs12509517 (G>C).Among them, a common functional insertion/deletion polymorphism (-94 insertion/deletion ATTG, rs28362491), located between 2 putative key promoter regulatory elements, is the most frequently investigated genetic variant. 4][25][26][27][28][29][30][31][32][33][34] A stratified meta-analysis revealed that this polymorphism can exert ethnic-and cancer-specific effects on cancer risk, [28][29][30][31][32][33][34] but to fully establish these differences, further large-scale and functional studies are necessary.There are several factors that may explain the occurrence of discrepancies in the results of the above studies, the most important of which are small sample sizes and diverse ethnicity, which affect the distribution of polymorphisms and environmental factors.This genetic variant, consisting of the deletion of 4 nucleotides (ATTG) in the promoter region of the NF-κB1 gene, reduces or completely prevents binding to nuclear proteins and leads to a reduction in the level of the NF-κB1 gene transcript, thereby altering mRNA stability and regulating translation efficiency. 11In bladder cancer, the presence of the del allele (ins/del + del/del genotype) reduces NF-κB1 (p50) mRNA expression in tumor tissues. 26Moreover, patients homozygous for the deletion possess a statistically higher risk of tumor recurrence than carriers with 1 or more insertion alleles in non-muscle invasive bladder cancer. 26rior to this study, the potential role of the NF-κB1 -94 ins/del ATTG (rs28362491) genetic polymorphism in the development and course of chronic lymphocytic leukemia had not been investigated.We have shown for the first time a significant impact of this genetic variation on CLL pathogenesis.A recessive polymorphic homozygous NF-κB1 -94 del/del ATTG variant significantly modified the risk of CLL in our Polish CLL patients, increasing the disease risk 2.23-fold.Additionally, the CLL-prone recessive del/del genotype was the most frequent variant in CLL patients with Rai stage 0, so the del/del-positive patients more frequently presented with the less aggressive disease phenotype, suggesting a low probability of progression to more advanced disease.Although this is a variant associated with increased risk of CLL disease, it may also be associated with the maintenance of the disease in the early, mildest stage. The enhanced expression and activity of p50 (NF-κB1) 20,36,37 as a result of this polymorphism may serve as an explanation for this phenomenon.The del allele is reportedly associated with the decreased promoter activity and enhanced NF-κB1 mRNA expression, 20,36,37 which might influence cancer development.As a transcription factor NF-κB component, p50 (NF-κB1) serves an important function in inhibiting cell apoptosis by modulating the expression levels of several survival genes (Bcl-2 homologue A1, PAI-2 and IAP gene family), which reveal the contribution of p50 (NF-κB1) signaling pathways to cellular proliferation by increasing IL-5, promoting MAPK phosphorylation and modulating cyclin D1 expression. 38Consequently, the observed association between the NF-κB1 -94 ins/del ATTG polymorphism and cancer risk can be explained by decoupling this genetic variation from its roles in apoptosis and cellular proliferation by modulating the expression of p50 (NF-κB1), 19,36,37 which is implicated through the mechanism described above. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Our study has several limitations, including the study size, and further, well-designed studies with representative sample sizes are necessary to validate our findings.A comparative analysis of age, sex and other prognostic factors such as the mutation status of the gene encoding immunoglobulin heavy chains should be taken into account in order to prove that the NF-κB1 promoter -94 ins/del ATTG polymorphism is a significant CLL risk and modulatory factor.Therefore, firstly the relevant studies should be developed by increasing the sample size in both study groups.Second, comparative analysis of age, sex and other prognostic factors, such as mutation status of the gene encoding immunoglobulin heavy chains, should be considered. Finally, we cannot exclude the possibility that the ATTG polymorphism of the NF-κB1 -94ins/del promoter may be an important factor of both risk and modulating CLL; therefore, further well-designed studies with a representative sample size are needed to definitively elucidate the role of this polymorphism to confirm our findings. ", "section_name": "Limitations", "section_num": null }, { "section_content": "We suggest that the del/del polymorphism genotype -94 ins/del ATTG (rs28362491) in the NF-κB1 gene increases the risk of chronic lymphocytic leukemia.This may be associated with retardation of disease progression and maintenance at an early, mild stage.In populations with increased frequencies of wild-type (insertion) alleles for -94 ins/del ATTG polymorphism (rs28362491) in the NF-κB1 gene, the probability of disease progression may be increased. ", "section_name": "Conclusions", "section_num": null } ]	[]
10.3389/fonc.2024.1490202	Review of BCL2 inhibitors for the treatment of Waldenström’s macroglobulinaemia and non-IgM lymphoplasmacytic lymphoma	<jats:p>Lymphoplasmacytic lymphoma (LPL) is a relatively rare form of indolent B-cell non-Hodgkin’s lymphoma, termed Waldenström’s macroglobulinaemia (WM) in the presence of an IgM paraprotein. Although traditionally treated with combination chemoimmunotherapy, the management is evolving in the era of targeted molecular therapies including Bruton’s tyrosine kinase inhibitors (BTKi). However, intolerance and refractoriness to BTKi mean newer agents are required, and the prognosis of so-called quadruple-refractory patients is poor. BCL2 is an anti-apoptotic, pro-survival protein that promotes lymphoma cell survival. Inhibition of BCL2 using first-in-class agent venetoclax has already altered the treatment paradigm in other conditions, including chronic lymphocytic leukaemia (CLL). <jats:italic>In-vivo</jats:italic> inhibition of BCL2 has been shown to lead to apoptosis of LPL/WM cells. Five studies have published results on the use of BCL2 inhibitors in WM to date, including oblimersen sodium, venetoclax, and sonrotoclax. Fixed-duration venetoclax resulted in high response rates, but many patients relapsed following the completion of therapy. The combination of venetoclax with ibrutinib resulted in higher and relatively deep response rates, but unexpected deaths due to ventricular events mean this combination cannot be explored. Two pivotal trials are currently evaluating the use of fixed-duration venetoclax, either in combination with rituximab or pirtobrutinib, whereas another multi-arm study is studying the use of continuous sonrotoclax monotherapy for R/R WM or in fixed-duration combination with Zanubrutinib for treatment-naïve patients. The potential role of BCL2 inhibitors in WM/LPL remains under study, with many hopeful that they may provide an additional chemotherapy-free oral alternative for patients requiring treatment. In an indolent condition with existing effective treatment regimens, including CIT and cBTKi, cost-effectiveness and toxicity profile will be key, although an additional treatment modality for quadruple-refractory patients with limited treatment options is urgently required.</jats:p>	[ { "section_content": "Lymphoplasmacytic lymphoma (LPL) is a relatively rare form of indolent B-cell non-Hodgkin's lymphoma and a distinct pathophysiological disease entity (1).In the great majority of patients, the lymphoma secretes a monoclonal immunoglobulin M (IgM), and the disease is eponymously termed Waldenström's macroglobulinaemia (WM).There is an additional recognised subtype, termed non-WM-type LPL that represents approximately 5% of cases and includes those with non-secretory disease, IgG or IgA paraproteins, and technically IgM LPL without bone marrow involvement (2).All are indolent but ultimately incurable conditions.The median age at diagnosis is approximately 70 years, and there is a 2:1 male preponderance.The incidence is estimated at 0.38 per 100,000 persons per year (3). 2 Pathophysiology of LPL/WM WM/LPL arises through the neoplastic proliferation of terminal B cells, which retain the ability to undergo plasmacytic differentiation.This results in an infiltrate of both clonal B and plasma cells in varying proportions.There is a precursor phase, termed monoclonal gammopathy of undetermined significance (MGUS), which is followed by progression to asymptomatic and finally symptomatic LPL/WM.During this process, there is gradual accumulation of LPL with clonal and subclonal molecular evolution.Gradual infiltration in the bone marrow results in cytopaenias, most commonly anemia, but infiltration of other tissues can also occur, including lymph nodes, spleen, bones, and the central nervous system. Serum IgM concentration appears to correlate with the degree of plasmacytic differentiation of the lymphoma, rather than simply overall disease burden (4).High levels of monoclonal IgM in the plasma result in symptoms of hyperviscosity and acquired von Willebrand syndrome, owing to the immunoglobulin's large pentameric and hexameric configuration (5).Monoclonal IgM can also cause autoimmune phenomena according to its antigenic target, resulting in complement-mediated destruction, such cold agglutinin syndrome (CAS) and anti-myelin-associatedglycoprotein (MAG) neuropathy.Precipitation of IgM in the form of types I and II cryoglobulin is seen, resulting in cryoglobulinaemic vasculitis (6). Transformation to a more aggressive, high-grade B-cell lymphoma occurs in up to 4% of patients and has a more adverse prognosis (7). ", "section_name": "Introduction", "section_num": "1" }, { "section_content": "Recurring somatic mutations have been identified in LPL cells (8).The activating single-point mutation L265P in the gene myeloid differentiation primary response 88 (MYD88) is present in >90% of patients with WM and non-IgM LPL.MYD88 L265P complexes with downstream effectors of B cell receptor signalling, such as IRAK1 and 4, leading to increased activation of the NFkB pathway.In addition, mutated MYD88 causes hyperactivation of haematopoetic cell kinase (HCK), in turn inducing PI3K/AKT, MAPK/ERK, and BTK signalling (9).These pathways drive cell survival and proliferation.Other MYD88 point mutations have also been described, including S219C, M232T, and S243N (10).MYD88 mutations are absent in patients with IgM multiple myeloma, a wholly separate disease entity characterised by t (11;14) (11). Additionally, WHIM-like mutations in the gene C-X-C chemokine receptor type 4 (CXCR4) are seen in approximately 30%-40% of cases of LPL/WM.These are subclonal and acquired after MYD88-mutations in disease pathogenesis (10,12).Half are CXCR4 S338X , but >40 frameshift or nonsense mutations are described (13).These mutations result in a CXR4 protein resistant to intracellular inactivation, thereby driving AKT, ERK, and MAPK1/2 pathway signaling, again promoting cellular survival.These mutations are associated with a degree of resistance to BKTi (14), higher serum IgM, and increased incidence of hyperviscosity and acquired von Willebrand syndrome (15, 16). Other, uncommonly identified somatic mutations include in AT-rich interactive domain 1A (ARID1A), cluster of differentiation (CD)79B, and lysine methyltransferase 2D (KMT2D) (8,17,18).TP53 aberrations are seen in <10% of patients, associated with resistance to chemotherapy and shorter overall survival (18)(19)(20)(21).Most cases of WM also feature multiple chromosomal abnormalities, most frequently deletion 6q, which is associated with progression to symptomatic disease (22,23). ", "section_name": "Disease genomics", "section_num": "2.1" }, { "section_content": "Treatment for WM/LPL is instituted when there are complications or symptoms related to the disease.Many patients can initially be monitored following the diagnosis, in the absence of disease-related symptoms or organ compromise, but the majority come to require therapy (24).The disease is ultimately incurable, but remissions lasting several years following treatment are often seen (25).There is international heterogeneity in the choice of anticancer therapy regimen, according to patient age, fitness, comorbidities, disease burden, genomic findings, and local health system reimbursement arrangements. ", "section_name": "Current therapeutic approach", "section_num": "3" }, { "section_content": "Combination chemoimmunotherapy (CIT) with an anti-CD20 monoclonal antibody (typically rituximab) remains the cornerstone of frontline therapy.Cytotoxic agents, such as purine analogue bendamustine (BR), alkylator cyclophosphamide with dexamethasone (DRC), and the proteasome inhibitor (PI) bortezomib with dexamethasone and cyclophosphamide (B-DRC) are widely used with good efficacy (26,27).BR results in 2-year overall survival (OS) of 97% and progression-free survival (PFS) 89%, DRC 91%, and 69%, and B-DRC 94 and 81%, respectively (28-30). ", "section_name": "Combination chemoimmunotherapy", "section_num": "3.1" }, { "section_content": "Targeted agents are dramatically changing the treatment landscape, particularly in the relapsed and refractory (R/R) setting.Following the discovery of MYD88 L265P , the use of targeted covalent BTK inhibitors (cBTKi) was shown to lead to apoptosis of WM cells (31,32) leading to successful clinical trials (33).cBTKis are now commonly employed at relapse and continued until disease progression.In some healthcare systems, it is also available for frontline use without prior exposure to CIT and may be preferable to CIT in those with significant comorbidities or frailty (27).First-generation cBTKi ibrutinib demonstrated a 90.5% ORR and 79.4% MRR in R/R WM, with 5-year OS 87% and PFS 54% (34). Subsequent iterations of cBKTis include next-generation agents Zanubrutinib and Acalabrutinib.Ibrutinib and Zanubrutinib have received Food and Drug Administration and European Medicines Agency approvals for use in WM, whereas Acalabrutinib is not available in Europe for this indication.There was no statistically significant difference in response rates between Ibrutinib and Zanubrutinib in the randomised phase III clinical trial ASPEN (94% vs. 95%, respectively) for R/R WM, with a median duration of response not reached at 44 months follow-up.The observed side effect profile was generally more favourable with Zanubrutinib (35).Acalabrutinib demonstrated efficacy in WM in a single-arm phase II study, with a 93% ORR of 93% both frontline and in R/R WM. cBTKi orelabrutinib and tirabrutinib have also demonstrated efficacy in Chinese and Japanese multicentre studies (36,37). However, complete responses are not attained with cBTKi and drug discontinuation often leads to rapid disease flare, necessitating continuous therapy until progression (38).Disease progression and resistance to cBTKi occur through several mechanisms.Half of cases due to the subclonal mutation BTK C481S , affecting the drug binding site, as well mutations in the downstream protein PLCg2 (39).Patients who are quadruple-agent refractory (i.e., to an alkylating agent, rituximab, cBKTi, and PI) have a poor prognosis, with a median OS of 13.2 months (38). Novel targeted agents under investigation in clinical trials include BCL2 inhibitors (BCL2i), non-covalent BTKi (ncBTKi) [such as nemtabrutinib and pritobrutinib (40,41)], BTK degraders, and cellular therapies with bispecific T-cell engagers (such as epcoritamab) and chimeric antigen receptor T-cell (CAR-T) therapy (42,43).This review will focus on the role of BCL2 inhibitors. ", "section_name": "Bruton's tyrosine kinase inhibitors", "section_num": "3.2" }, { "section_content": "The B-cell lymphoma 2 (Bcl-2) family of proteins has long been known to be key cellular apoptotic regulators (44), and the relationship between its pro-and anti-apoptotic members largely determines whether a cell survives or dies (45).Bcl-2 is the name of one of the constituent anti-apoptotic, pro-survival proteins within the wider Bcl2-group, which is capable of sequestering the pro-apoptotic proteins BAK, BAX, and other Bcl-2 homology domain 3 (BH3)-only proteins.The regulation of Bcl-2 family proteins is complex but ultimately results in a binary decision whether a cell lives or undergoes apoptosis. The therapeutic use of BCL2i has achieved impressive results in other B-cell disorders, notably chronic lymphocytic leukaemia (CLL), and drastically shifted the treatment landscape away from CIT (46).Gene expression profiling in LPL/WM cells has identified upregulation of BCL2, similar to that seen in CLL (47).In-vitro exposure of MYD88 MUT cells, both CXCR WT and CXCR WHIM , resulted in apoptosis and has paved the way for therapeutic studies (47). ", "section_name": "Rationale for BCL2 inhibitor use in LPL/WM", "section_num": "4" }, { "section_content": "No BCL2i is currently licenced for the treatment of WM/LPL.The only licensed BCL2i in haematological disorders is venetoclax, a small molecule that binds to BH3 and prevents the sequestering of anti-apoptotic proteins, thereby promoting apoptosis (48).It is licenced for and widely used in, CLL and acute myeloid leukaemia (AML).Several other BCL2 inhibitors are in development.Sonrotoclax is a second-generation, highly potent, and selective inhibitor of BCL2.It has shown greater in vitro inhibition of BCL2 than venetoclax, and currently undergoing testing in clinical trials.Interestingly, it has shown the ability to overcome several common venetoclax-resistance mutations, such as G101V in pre-clinical mouse models (49,50). To date, five studies have evaluated the use of BCL2i in LPL/ WM (Table 1): • The first was a phase 1/2 multicentre dose-escalation trial (NCT00062244), by Gertz and colleagues at the Mayo clinic, in patients with WM who received oblimersen sodium.This was an antisense oligonucleotide for the first six codons of the BCL2 open reading frame, which prevents the expression of the gene product.The results of the phase 1 portion were published in 2005, with a partial response in one of nine enrolled patients, but with grade 3 or higher haematological toxicities in five patients (51).A total of 58 patients were enrolled between 2003 and 2007, but no formal results of the phase 2 portion have been published to date (52).(16).A total of 45 patients were recruited between 2020 and 2022.MYD88 WT patients were excluded.Treatment cycles were administered every 28 days.Cycle one consisted of ibrutinib 420 mg, with the addition of venetoclax from cycle 2. Venetoclax was administered once daily at 100 mg for 1 week, 200 mg for another week, and 400 mg for 2 weeks.From cycles 3 to 24, participants received ibrutinib 420 mg and venetoclax 400 mg once daily, unless there was disease progression or unacceptable toxicity.TLS prophylaxis was provided in the form of outpatient oral hydration and allopurinol.The ORR was 100% and MRR 96%, with 42% attaining a VGPR, 53% PR, and 4% MR.The 24-month OS rate was 96% and PFS 76%.Crucially, ventricular arrhythmias occurred in three patients, with two deaths and one grade 4 event.The affected patients were all male, >65 years, and had cardiac comorbidities including a history of arrhythmia, coronary artery disease, hypertension, hypercholesterolemia, diabetes, or obesity. The study therapy was subsequently terminated after a grade 2 ventricular arrhythmia occurred in a participant undergoing a precautionary cardiac stress test, resulting in a concerning overall rate of ventricular arrhythmias of 9% in this study.Neutropaenia was common, including 17/45 grade ≥3 neutropaenias.The study authors concluded that there is a likely additive effect of combining BTK and BCL2 inhibitors in WM, with high rates of VGPR, rapid responses, and significant reduction in bone marrow burden (decreasing from 60% to 5% at best response), but that there was an unacceptably high incidence of ventricular events, including two deaths.Intriguingly, such events were not seen in other trials using ibrutinib and venetoclax combinations for other B-cell neoplasms (55,56).Possible explanations include undetected cardiac involvement by AL amyloid, other cardiac paraprotein deposition, or an inherently higher risk of cardiac events in the patient cohort due to co-morbidities.The combination of ibrutinib and venetoclax for WM/LPL is therefore not recommended or currently planned for further study (16).• A phase 1a/1b open-label dose escalation and expansion study is examining the use of second-generation sonrotoclax in patients with mature B-cell malignancies (NCT04277637) (57).Interim results were reported on a dedicated cohort of 17 patients with R/R WM, enrolled into three dose escalation cohorts.Previous cBTKi-exposure was noted in 10 patients.At a median follow-up of 10.6 months, four (24%) patients had progressed and two had discontinued due to adverse events.ORR was 76%, MRR 41%, and VGPR 12%.The study aims to complete by 2027. ", "section_name": "Existing evidence for BCL2 inhibitors in LPL/WM", "section_num": "5" }, { "section_content": "There are currently three trials in progress, evaluating the use of BCLi in LPL/WM (Table 2): There are three cohorts: cohort 1 is for participants with R/ R disease to both cBTKi and anti-CD20-containing CIT, cohort 2 R/R disease to anti-CD20-containing CIT with intolerance to BTKi, and cohort 3 R/R disease to BTKi and unsuitable for CIT.In addition, a fourth subcohort will evaluate the use of sonrotoclax in combination with cBTKi zanubrutinib in treatment-naïve patients for a fixed duration.The primary outcome measure is MRR, with secondary outcomes including ORR, rates of ≥VGPR, duration of response, PFS, and OS (60). ", "section_name": "Trials in progress for BCL2 inhibitors in LPL/WM", "section_num": "6" }, { "section_content": "WM/LPL is an indolent but incurable condition, typically requiring several lines of therapy punctuated by periods of relative remission.Although CIT continues to be used, particularly for treatment-naïve patients where it has demonstrated good response rates and PFS, cBTKis are the mainstay of R/R WM.With increasing duration of use, resistance to cBTKis is an emerging problem and other oral agents are needed.A proportion of patients are also unable to tolerate BTKis due to class-specific side effects, such as bleeding.In addition, the prospect of a \"chemotherapy-free,\" fixed-duration frontline therapy would no doubt be appealing to patients and clinicians. A total of five studies have examined the use of BCL2 inhibitors in WM/LPL: one oblimersen sodium, three venetoclax, and one sonrotoclax.Three pivotal trials are currently in progress, with the potential to alter practice in the treatment of LPL/WM.Fixedduration venetoclax monotherapy for 24 months resulted in highresponse rates but significant progression within 12 months of completion.This suggests that either continuous or combination therapy is required for the use of BCL2i in this condition.The combination of a BTKi ibrutinib and BCL2i venetoclax in LPL/WM, unfortunately, resulted in unacceptably high rates of ventricular events and death (16), essentially excluding this drug combination from further evaluation in WM/LPL.A cautious interpretation of the rapid responses and high rates of VGPR with this combination suggests that BCL2i in combination with other covalent-and noncovalent BTKis, with less cardiotoxic side effect profiles and in selected patients, may be of interest.The combination may particularly have a role in the context of heavy bone marrow infiltration, given the high rates of bone marrow clearance seen.This combination is currently being evaluated in two studies, one using sonrotoclax-zanubrutinib frontline and another using venetoclax-pirtobrutinib for R/R WM.The combination of a BCL2i with an anti-CD20 antibody, such as rituximab, may also prove efficacious. The role of BCL2 inhibitors in WM/LPL remains under study, with many hopeful that they may provide an additional chemotherapy-free oral alternative for patients requiring treatment, both frontline and in the R/R setting.In an indolent condition with existing effective options, including CIT and cBTKi, costeffectiveness and toxicity profile will be key.However, they are likely to find an important role if capable of offering an effective therapeutic mechanism for patients' refractory to existing therapeutic options with limited options, particularly quadruple refractory patients. ", "section_name": "Conclusion", "section_num": "7" } ]	[ { "section_content": "The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article. ", "section_name": "Funding", "section_num": null }, { "section_content": "OT: Writingoriginal draft, Writingreview & editing.SD'S: Writingreview & editing. SD'S has received research support from Janssen and BeiGene, and advisory board honoraria from Janssen, BeiGene, HillStarBio, Kite and Sanofi. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The author(s) declared that they were an editorial board member of Frontiers, at the time of submission.This had no impact on the peer review process and the final decision. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. ", "section_name": "Author contributions", "section_num": null }, { "section_content": "OT: Writingoriginal draft, Writingreview & editing.SD'S: Writingreview & editing. ", "section_name": "Author contributions", "section_num": null }, { "section_content": "SD'S has received research support from Janssen and BeiGene, and advisory board honoraria from Janssen, BeiGene, HillStarBio, Kite and Sanofi. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The author(s) declared that they were an editorial board member of Frontiers, at the time of submission.This had no impact on the peer review process and the final decision. ", "section_name": "Conflict of interest", "section_num": null }, { "section_content": "All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. ", "section_name": "Publisher's note", "section_num": null } ]
10.1186/s40164-022-00302-0	CircRIC8B regulates the lipid metabolism of chronic lymphocytic leukemia through miR199b-5p/LPL axis	<jats:title>Abstract</jats:title><jats:sec> <jats:title>Objective</jats:title> <jats:p>Circular RNAs (circRNAs) play a critical role in the modulation of tumor metabolism. However, the expression patterns and metabolic function of circRNAs in chronic lymphocytic leukemia (CLL) remain largely unknown. This study aimed to elucidate the role of circRNAs in the lipid metabolism of CLL.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>The expression and metabolic patterns of circRNAs in a cohort of 53 patients with CLL were investigated using whole transcriptome sequencing. Cell viability, liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis, lipid analysis, Nile red staining as well as triglyceride (TG) assay were used to evaluate the biological function of circRIC8B in CLL. The regulatory mechanisms of circRIC8B/miR-199b-5p/lipoprotein lipase (LPL) axis were explored by luciferase assay, RNA immunoprecipitation (RIP), qRT-PCR, and fluorescence in situ hybridization (FISH). CCK-8 and flow cytometry were used to verify the inhibition role of cholesterol absorption inhibitor, ezetimibe, in CLL cells.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>Increased circRIC8B expression was positively correlated with advanced progression and poor prognosis. Knockdown of circRIC8B significantly suppressed the proliferation and lipid accumulation of CLL cells. In contrast, the upregulation of circRIC8B exerted opposite effects. Mechanistically, circRIC8B acted as a sponge of miR-199b-5p and prevented it from decreasing the level of LPL mRNA, and this promotes lipid metabolism alteration and facilitates the progression of CLL. What’s more, ezetimibe suppressed the expression of LPL mRNA and inhibited the growth of CLL cells.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>In this study, the expressional and metabolic patterns of circRNAs in CLL was illustrated for the 1st time. Our findings revealed that circRIC8B regulates the lipid metabolism abnormalities in and development of CLL through the miR-199b-5p/LPL axis. CircRIC8B may serve as a promising prognostic marker and therapeutic target, which enhances the sensitivity to ezetimibe in CLL.</jats:p> </jats:sec>	[ { "section_content": "Chronic lymphocytic leukemia (CLL), which is particularly prevalent in Western countries, has shown an increasing incidence rate in China [1][2][3].Characterized by marked clinical heterogeneity, approximately 1/3 of patients with CLL experience a rapidly progressive ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Open Access", "section_num": null }, { "section_content": "clinical course [4].Despite extensive research and progress, therapies for CLL remain inadequate.Novel biomarkers for the prediction of CLL prognosis and further elucidation of the precise molecular mechanisms can improve the outcome of CLL patients.Accumulating evidence has illustrated the pivotal role of metabolism in tumors [5][6][7], and metabolism pattern is dictated by a variety of intrinsic and extrinsic factors.Non-coding RNAs (ncRNAs), including circular RNAs (circRNAs) was reported to influence tumor metabolism [8].For example, circACC1 contributes to metabolic adaptation and acts as a tumor promoter in colorectal cancer [9], and circRNA SCAR could regulate the metabolic inflammation of nonalcoholic steatohepatitis [10].In order to satisfy the energy demand, CLL cells could store lipids and utilize free fatty acids (FFAs) [11].Lipoprotein lipase (LPL) was found to be a key factor in the metabolism of CLL cells by promoting cellular uptake of lipoproteins and accelerating the hydrolysis of triglycerides (TGs) into FFAs.Ezetimibe, a FDA-approved drug could significantly reduce the levels of low-density lipoprotein cholesterol (LDL-C) and TGs by inhibiting intestinal absorption of cholesterol [12].Numerous studies claimed that CLL patients with higher LPL levels were usually correlated with aggressive disease and unfavorable prognosis [13,14].However, the role of circRNAs in mediating the metabolism of CLL cells, particularly in lipid metabolism, has not yet been reported.Therefore, we attempted to identify the metabolism-associated circRNA profiles in CLL and explored the underlying molecular mechanisms. Here, we identified circRIC8B, which promotes cell proliferation and lipid metabolism and is associated with poor outcomes in patients with CLL.Additionally, circRIC8B functions as an oncogenic driver to promote lipid metabolism by sponging miR-199b-5p, resulting in the upregulation of LPL mRNA.For the 1st time, our study explored the relationship between circRNAs and metabolism in CLL.These findings also provide new evidence that circRNAs function in CLL and offer a promising therapeutic target, broadening treatment options that targeting metabolism. ", "section_name": "Experimental Hematology & Oncology", "section_num": null }, { "section_content": "", "section_name": "Materials and methods", "section_num": null }, { "section_content": "Peripheral blood samples were obtained from previously untreated patients with CLL who were followed up at the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital.Written informed consent was obtained from the patients and use of human samples was approved by the ethics committee (Approval Number 2021-SRFA-208).Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep ™ (Stemcell Technologies, Vancouver, Canada) and stored in TRIzol at-80 °C until use. ", "section_name": "Human samples", "section_num": null }, { "section_content": "CLL cell lines MEC-1 and JVM-3 (X-Y Biotechnology, Shanghai, China) and Human B lymphocyte cell line GM12878 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS) (Yeasen, Shanghai, China) and 100 μg/mL penicillin/ streptomycin (Gibco).Human renal epithelial carcinoma HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco).The cell lines used in this study were authenticated by short tandem repeat (STR) profiling and maintained at 37 °C in a humidified atmosphere with 5% CO 2 . ", "section_name": "Cell culture", "section_num": null }, { "section_content": "Transient transfection with small interfering RNAs (siR-NAs) specifically targeting circRIC8B that synthesized by Geenseed Biotech (Guangzhou, China) was performed by using Lipofectamine ® RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer's protocol.Briefly, six thousand cells washed twice with sterile PBS were seeded in a 6-well plate.100 nM siRNAs and the same volume of RNAiMAX were diluted with OPTI-MEM medium separately and mixed and incubated for 20 min before added into cells.For transient transfection, cells are typically assayed 48 h after transfection.To generate CLL cell clines that stably overexpress circRIC8B, MEC-1 and JVM-3 were infected with lentivirus vectors (Geenseed Biotech) according to the manufacturer's protocol.After 3 days of infection, cells were selected with puromycin (2 µg/ml) for 3 days. ", "section_name": "Cell transfection", "section_num": null }, { "section_content": "Total RNA was extracted using TRIzol reagent according to the manufacturer's protocol.For RNase R treatment, 2.5 μg of total RNA was incubated with or without 3 U/μg of RNase R for 20 min at 37 °C (Epicentre Technologies, Madison, WI). cDNA was synthesized using a reverse transcription kit (Vazyme, Nanjing, China).For actinomycin D treatment, the culture medium was added with actinomycin D (2 ug/mL) and cells were collected at a specified time to assess the stability of circRNA.The separation of nuclear and cytoplasmic fractions of cells were performed using PARIS ™ Kit (Life Technologies).qRT-PCR was performed with SYBR Green Master Mix (Vazyme, Nanjing, China).The specific primers used are listed in Additional file 1: Table S1.GAPDH or U6 was used as an internal standard and relative RNA expression levels were calculated using the 2 -ΔΔCT method (See Additional file 1: Table S1). ", "section_name": "RNA extraction and quantitative real-time PCR", "section_num": null }, { "section_content": "For cell proliferation analysis, Cell Counting Kit-8 (CCK-8; APExBIO, Houston, TX, USA) was used.Briefly, 10 000 cells were seeded in 96-well plates and ten microliters of CCK-8 assay solution were added to each well at the indicated times.After 2 h's incubation, absorbance at 450 nm was measured.For growth inhibition analysis, cells were incubated with the indicated concentrations of ezetimibe (Selleck Chemicals, S1655) range from 0 to 40 μM for 48 h. ", "section_name": "Cell proliferation and growth inhibition assay", "section_num": null }, { "section_content": "For the cell apoptosis assay, cells were firstly washed twice with cold PBS and resuspended with binding buffer.Apoptotic levels of cells were quantified by flow cytometry (BD Biosciences) after 15 min's incubation at 4 °C with Annexin V and PI.Data analysis was performed using the CytExpert software. ", "section_name": "Flow cytometry", "section_num": null }, { "section_content": "The FISH assay was performed to observe the location of circRIC8B and miR-199b-5p in CLL cells.Cy3-labeled circRIC8B probes and 5FAM-labeled miR-199b-5p were designed and synthesized by Ribo-Bio (Guangzhou, China).FISH analysis was performed using a fluorescent in situ hybridization kit (RiboBio) according to the manufacturer's instructions.Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Beyotime, China).Images were obtained using a fluorescence microscope. ", "section_name": "Fluorescence in situ hybridization (FISH)", "section_num": null }, { "section_content": "The RIP assay was performed using the Magna RIP ™ RNA-binding protein immunoprecipitation kit (Millipore, USA) following the manufacturer's protocol.Briefly, 6 × 10 7 cells were collected and resuspended in 300 μL complete RIP lysis buffer.The cell lysates were incubated with antibodies against Argonaute-2 (AGO2; Abcam, MA, USA) or rabbit IgG and pre-treated magnetic beads at 4 °C overnight with rotation.Immunoprecipitated RNA was then reverse transcripted after purification.The enrichment of circRIC8B was evaluated using qRT-PCR. ", "section_name": "RNA immunoprecipitation (RIP)", "section_num": null }, { "section_content": "Intracellular lipid droplet analysis was performed using Nile red staining.Cells were treated with 1 mM FFA and fixed with 4% paraformaldehyde for 15 min.After staining with Nile red dye (1 μg/mL), the images were acquired using a fluorescence microscope.TG content in CLL cells was quantified using a triglyceride quantification kit (BioAssay Systems, ETGA-200) according to the manufacturer's instructions. ", "section_name": "Nile red staining and triglyceride (TG) assay", "section_num": null }, { "section_content": "HEK293 T cells were seeded in 24-well plates at 30% confluency for 24 h before transfection.The cells were cotransfected with a mixture of luciferase reporter vectors (pmirGLO) containing circRIC8B-miRNA binding or mutant sequences and miRNA mimics to examine their miRNA binding ability.After 48 h, luciferase activity was measured using a dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer's protocol and a previous study [15]. ", "section_name": "Dual-Luciferase reporter assay", "section_num": null }, { "section_content": "Total RNA was extracted from patients with CLL, followed by rRNA depletion.Then, the RNA was reverse transcribed to cDNA and constructed into a strandspecific library after purification by rRNA depletion.Sequencing was performed on an Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA).All raw data is accessible through the NCBI GEO database (SRA: PRJNA762572).GSVA was used to obtain the metabolic score and the subsequent metabolic heatmap [16]. ", "section_name": "Whole transcriptome sequencing", "section_num": null }, { "section_content": "A total of 10 7 cells were collected and washed twice with PBS.For metabolite extraction, an extraction solution (acetonitrile: methanol: water = 2: 2: 1) containing an isotopically labeled internal standard mixture was added to the sample.After a 30 s vortex, the samples were frozen and thawed with liquid nitrogen 3 times and then sonicated for 10 min in an ice-water bath.Supernatant was transferred to a fresh glass vial for LC-MS/MS analysis after centrifugation.Quality control (QC) samples were prepared by mixing an equal aliquot of the supernatant from all samples.LC-MS/MS analyses were performed using a UHPLC system (Vanquish, Thermo Fisher Scientific).Raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program, developed using R and based on XCMS, for peak detection, extraction, alignment, and integration.An in-house MS2 database (BiotreeDB) was then used for metabolite annotation.The cutoff for annotation was set at 0.3.Detection and analysis were provided by SHANG-HAI BIOTREE BIOTECH CO., LTD. ", "section_name": "Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analyses", "section_num": null }, { "section_content": "Graphpad Prism software version 8 was used to statistically analyze the data.All of the experiments were conducted in triplicate.P values were calculated using a Student's t-test.Spearman's rank correlation coefficient with t-test was used to evaluate correlative studies.Time to first treatment (TTT) was calculated from the date of diagnosis to the date of first treatment or last followup, considering disease-unrelated deaths as competing events.The Kaplan-Meier method and log-rank test were used to assess overall survival, and the Gray test was used to compare the cumulative incidence curves of TTT.Multivariate analyses of prognostic factors were performed using the Cox regression models.Statistical significance was set at p < 0.05. ", "section_name": "Statistical analyses", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "In our previous study, we elucidated the expression profile of circRNAs in the plasma of patients with CLL [15].To further explore the profile of cells from CLL patients, 53 treatment-naïve patients with CLL were enrolled.Whole transcriptome sequencing showed that circR-NAs were broadly expressed and at least 4000 circRNAs were captured in the majority of samples (Fig. 1A).These circRNAs were found generated from various genomic regions, including untranslated regions (UTRs), intergenic regions, introns, and most commonly protein-coding exons (Fig. 1B).In addition, the density distribution of circRNAs on different chromosomes are displayed in Fig. 1C.As most circRNAs are emanated from protein-coding genes, we then wondered the relationship between the abundance of circRNAs and their host genes.Host genes with higher mRNA levels tend to transcribe circRNAs (Fig. 1D).To further elucidate the correlation between host genes and circRNAs, host genes were divided into three groups according to their abundance.With a higher expression of host genes, circRNAs were likely to exhibit higher abundance (Fig. 1E).Moreover, our results indicated that the host genes with lower abundance of mRNAs showed a stronger positive correlation with the expression of their corresponding circRNAs (Fig. 1F).Collectively, these results illustrated that circR-NAs are highly and broadly expressed in CLL patients. ", "section_name": "Properties of circRNAs in CLL", "section_num": null }, { "section_content": "To mine the circRNAs that regulating metabolism, the scores of various metabolic pathways in patients with CLL were firstly calculated using the mRNA sequencing data (Additioinal file 2: Table S2).In addition, the heatmap exhibited the enrichment of metabolism pathways of individual patient (Fig. 2A), and relevant circR-NAs were shown in Fig. 2B.Given the significance of LPL in lipid metabolism, circRIC8B with proper length and relative high abundance, among circRNAs that positively correlated with LPL was selected for further study.Participant characteristics were collected and summarized (Fig. 2C).The expression levels of circRIC8B were observed significantly correlated with CLL patients' elevated low-density lipoprotein cholesterol (LDL-C) levels.In addition, patients with higher circRIC8B levels were accompanied with more, advanced Rai stage, and unmutated immunoglobulin heavy-chain variable region (IGHV) status.Meanwhile, LPL was observed positively associated with circRIC8B expression (Additional file 3: Table S3).Gene set enrichment analysis (GESA) of associated genes was also performed, and the results revealed that circRIC8B was involved in metabolism, including fatty acid metabolism (Fig. 2D,E). ", "section_name": "CircRNAs involved in the metabolism of CLL", "section_num": null }, { "section_content": "circRIC8B was generated from the head-to-tail splicing of exons 4 to 8 from RIC8B precursor-mRNA (pre-mRNA) (710 bp).Primers were designed according to the back-splicing junction and the head-to-tail splicing was confirmed with sanger sequencing (Fig. 3A).The RNase R digestion assay demonstrated that circRIC8B was resistant to RNase R, whereas the linear isoform was decreased after RNase R treatment (Fig. 3B).In addition, circRIC8B was more stable than RIC8B mRNA in CLL cells treated with actinomycin D, a transcription inhibitor (Fig. 3C).Nuclear and cytoplasmic fraction assays and fluorescence in situ hybridization (FISH) showed circRIC8B was predominantly distributed in cytoplasm (Fig. 3D,E).Collectively, these findings demonstrated that circRIC8B was an abundant, circular, and stable transcript that was expressed in CLL cells. ", "section_name": "Characterization of circRIC8B in CLL", "section_num": null }, { "section_content": "Further PBMC samples from 63 CLL patients were collected to detect the expression levels of circRIC8B and confirm its clinical significance.Kaplan-Meier analysis displayed that patients with higher circRIC8B expression had a worse prognosis and shorter survival time (Fig. 4A).Moreover, patients with CLL who had low levels of cir-cRIC8B had a notably longer time to first treatment time (TTT) (Fig. 4B).The results were the same as those for the LPL (Additional file 4: Fig S1A,B).In addition, the expression levels of circRIC8B and LPL mRNA were elevated in patients without IGHV mutation compared to those in the IGHV mutated patients (Fig. 4C, Additional file 4: Fig S1C).In line with the TCGA database [17], IGHV mutation status corresponded with the expression of LPL mRNA (Fig. 4C, Additional file 1: Fig S1D).The relationship between circRIC8B level and other clinicopathological characteristics was also evaluated (Fig. 4D).The expression of circRIC8B was found to be associated with IGHV mutation status rather than other clinical characteristics, including age, sex, cytogenetics, and stage (Table 1).To eliminate the possible contamination of .We further investigated the effects of circRIC8B on cell proliferation and small interfering RNAs (siRNAs) that specifically target circRIC8B and circRIC8B overexpression vector were constructed and validated by agarose gel electrophoresis and sanger sequencing (Additional file 6: Fig S3A andB).The knockdown and overexpression efficacy were validated by qRT-RCR (Fig. 4E andF, Additional file 6: Fig S3C andD).Cell counting kit-8 proliferation (CCK-8) assay showed that knockdown of circRIC8B notably suppressed the proliferation ability of CLL cells, whereas overexpression of circRIC8B led to an increase in cell viability (Fig. 4E-H).These data suggested that circRIC8B plays an oncogenic role in CLL. ", "section_name": "CircRIC8B predicts aggressive clinical characteristics in patients with CLL and promotes cell proliferation", "section_num": null }, { "section_content": "To determine the role of circRIC8B in metabolism, liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was performed with six biological replicates between cells with or without circRIC8B siRNA interference.The principal component analysis (PCA) score plot showed a clear separation between the si-circRIC8B and control groups (Fig. 5A).The identified metabolites were then used to perform supervised clustering, orthogonal partial least squares discriminant analysis (OPLS-DA) (Fig. 5B).The analysis produced a strong model with highly validated predictability and goodness of fit (Fig. 5C).Volcano plots showed that the metabolites were differentially expressed between the two groups (Fig. 5D, Additional file 7: Table S4).Metabolic pathway analysis was performed, and the lipid metabolism pathway was observed the most considerably altered between the two groups (Fig. 5E).This indicated that circRIC8B was a potential regulator that plays a key role in lipid metabolism.To test this hypothesis, Nile red staining was performed (Fig. 5F), quantitative analysis of mean fluorescence intensity (MFI) was performed (Fig. 5G).The results showed that lipid accumulation was increased in CLL cell lines with circRIC8B overexpression, whereas a significant decrease in lipid accumulation was observed in the circRIC8B knockdown cells.Besides, cells overexpressing circRIC8B exhibited a higher Triglyceride (TG) content, which is a quantitative indicator of lipid accumulation, whereas a lower TG content was detected in the circRIC8B knockdown cells, in line with the results of Nile red staining.Taken together, circRIC8B was regarded as a key factor that regulates lipid accumulation and then promoted cell proliferation in CLL (Fig. 5H,I). ", "section_name": "CircRIC8B regulates lipid metabolism in CLL", "section_num": null }, { "section_content": "Given that circRIC8B was distributed in the cytoplasm and positively correlated with LPL mRNA, we investigated whether circRIC8B acted as a microRNAs (miR-NAs) sponge in CLL cells.RNA immunoprecipitation (RIP) assay showed that circRIC8B was specifically enriched against Argonaute-2 (AGO2) (Additional file 8: Fig S4A).Then, we used circBank to predict miRNAs that bind circRIC8B and starBase to predict miRNAs that target LPL mRNA.The results showed that five miRNAs, including miR-199a/b, miR-27a/b, and miR-892c, overlapped (Fig. 6A).Notably, miR-199b-5p was the only one that was upregulated after transfection with circRIC8B siRNA (Fig. 6B).The FISH analysis confirmed the location of circRIC8B and miR-199b-5p mainly in the cytoplasm of CLL cell lines MEC-1 and JVM3, and importantly, they were co-localized (Fig. 6C).Dual-luciferase reporter assays were then performed.Compared with that in cells transfected with the mutant sequence, the luciferase reporter activity was significantly decreased by the miR-199b-5p mimics in cells transfected with the wildtype sequence (Fig. 6D).However, no significant change was observed when transfected with miR-199a, miR-27a/b, or miR-892c mimics (Additional file 8: Fig S4B).The change in LPL mRNA expression was verified after transfecting CLL cell lines with the miR-199b-5p mimic and inhibitor.LPL mRNA was downregulated with an increased level of miR-199b-5p and upregulated with a decreased miR-199b-5p (Fig. 6E-H).Luciferase reporter activity further confirmed the binding of miR-199b-5p and LPL mRNA (Fig. 6I).Additionally, LPL mRNA levels were significantly decreased after silencing circRIC8B (Fig. 6J).Collectively, our results provided evidence that circRIC8B can directly bind to miR-199b-5p and regulate the expression of LPL mRNA in CLL cells. ", "section_name": "CircRIC8B serves as a sponge of miR-199b-5p", "section_num": null }, { "section_content": "We then verified whether circRIC8B induced CLL progression through the circRIC8B/miR-199b-5p/LPL axis.MEC-1 and JVM-3 were transfected with circRIC8B siRNA and/or miR-199b-5p inhibitor, and CCK-8 assays showed that circRIC8B knockdown significantly suppressed cell proliferation, whereas miR-199b-5p inhibitor induced proliferation.Transfection with miR-199b-5p inhibitor reversed the decrease of cell viability (Fig. 7A,B) and the reduction of lipid accumulation caused by circRIC8B knockdown in CLL cell lines (Fig. 7C,D).As expected, the changes in TG content showed the same results as the Nile red staining assays (Fig. 7E).Next, we investigated the expression and correlation of circRIC8B, miR-199b-5p, and LPL mRNA using PBMC samples from 63 CLL patients.Pearson correlation analysis indicated that miR-199b-5p was negatively correlated with circRIC8B and LPL mRNA, whereas circRIC8B levels were positively correlated with LPL mRNA (Fig. 7F-H).Together, these results verified the function of cir-cRIC8B/miR-199b-5p/LPL mRNA axis in CLL. ", "section_name": "CircRIC8B functions through the miR-199b-5p/LPL axis in CLL", "section_num": null }, { "section_content": "CLL cell lines with ezetimibe and we observed cell growth inhibitory effects in a dose-dependent manner.Notably, the inhibitory effects were much more obvious in the stable cell lines transfected with sh-circRIC8B.However, there was no significant difference between the control groups and circRIC8B overexpression groups in ezetimibe sensitivity (Fig. 8A,B).A similar result was observed in the apoptotic analysis (Fig. 8C,B, Additional ", "section_name": "CircRIC8B knockdown enhances the sensitivity of CLL cell lines to ezetimibe, which inhibits cell viability and LPL mRNA expression", "section_num": null }, { "section_content": "In order to meet the increased demand for energy and metabolites owing to the rapid proliferation and survival of tumor cells, metabolic reprogramming was subsequently driven [18,19].CLL is a disease with approximately 1% of tumor cells proliferating every day, more than commonly thought [20].Although CLL cells maintain high levels of proliferation through metabolic changes, extensive studies have not clearly explained the underlying mechanism of driving genes in the progress [21].CircRNA is one of the types of non-coding RNAs, the novel functions and molecular mechanisms of which in hematological malignancies have attracted researchers' attention [22].Meanwhile, circRNAs were reported to exert vital functions in the metabolism of multiple tumors [23,24].However, the metabolic profile and role of circRNAs in the regulation of CLL metabolism remain largely unclear. We investigated the circRNA expression profile in PBMCs from 53 patients with CLL using whole transcriptome sequencing and analysed the circRNAs involved in metabolism pathways.A new circRNA termed circRIC8B was observed to be remarkably correlated with LPL and significantly associated with the poor prognosis of patients with CLL.In addition, cir-cRIC8B was a key factor in lipid accumulation through the miR-199b-5p/LPL axis, resulting in significant changes in cellular lipid storage, thus supporting the proliferation of CLL cells (Fig. 8I).Multiple reports showed that ncRNAs, such as lncRNAs, miRNAs, and circRNAs, contribute to tumor metabolism [24].For example, miR-199b-5p, recognized as a tumor-suppressive miRNA, is involved in the lipid metabolism of breast cancer [25].We have been dedicated to the study of circRNAs in CLL and reported that plasma circ-RPL15 serves as a diagnostic and prognostic marker and mitochondrial genome-derived circRNA mc-COX2 functions as an oncogene in CLL [15,26].To date, only a few circRNAs have been verified and explored in CLL [27,28].In the present study, we focused on the circR-NAs participated in the metabolism modulation.Dramatically, our findings revealed new clues for the role of circRNA in abnormal lipid metabolism in CLL. The estimation of tumor metabolism has a critical prognostic value in the evaluation of patients' responses to treatment [29].Targeting specific metabolism genes has shown clinical significance in the elimination and management of tumors [30,31].Increased LDL-C levels are strongly associated with an increased risk of coronary artery disease and other atherosclerosis-related disorders [32,33].Ezetimibe is a cholesterol absorption inhibitor that is reported to be an effective and generally well-tolerated option to reduce LDL-C concentration and confirmed to be effective and generally well-tolerated in patients [34].The addition of ezetimibe to statin therapy helps to reduce LDL-C concentration and provides additional cardiovascular benefits [35].Recent studies suggest there is a high incidence of elevated LDL levels in CLL patients.Thus, lipid lowering therapy may benefits CLL.Statin drugs reduce low-density lipoprotein (LDL)-cholesterol (LDL-C) and cardiovascular risk.Ezetimibe may be used to supplement statin therapy, or used alone in cases of statin intolerance [36,37].As LPL was the target gene of circRIC8B, which was positively correlated with LDL-C, and contributed to lipid accumulation, ezetimibe was then applied to assess its antitumor function.As expected, ezetimibe showed effective inhibitory effects on CLL cells by reducing the levels of LPL.Knockdown of circRIC8B significantly suppressed the lipid accumulation and proliferation of CLL cells.What's more, decreased expression of circRIC8B also enhanced their sensitivity to ezetimibe, which indicated that higher levels of circRIC8B may be the factor that blocked the effectiveness of ezetimibe.Meanwhile, the levels of circRIC8B were upregulated with ezetimibe treatment, which may be explained by a compensatory regulation underlying some other signaling pathways while this needs to be further illustrated in future study.These results indicated that circRIC8B contributes to the maintenance of CLL cells during the inhibition of lipid metabolism.Ezetimibe, which exerts potential anti-oncogenic effect may be implicated in lipid metabolism through diverse regulatory mechanisms from circRIC8B.Our findings indicated that targeting circRIC8B with the addition of metabolic inhibitors may be a potential therapeutic strategy for CLL. ", "section_name": "Discussion", "section_num": null }, { "section_content": "To our knowledge, this is the first study to thoroughly investigate the relationship between circRNAs and metabolism in hematological diseases with the largest cohort.Our data provides additional evidence for the functions of circRNA in lipid metabolism and CLL pathogenesis, which may broaden the research horizons. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year ", "section_name": "Conclusions", "section_num": null }, { "section_content": "At BMC, research is always in progress. ", "section_name": "•", "section_num": null }, { "section_content": "Ready to submit your research Ready to submit your research ?Choose BMC and benefit from: ? Choose BMC and benefit from: ", "section_name": "Learn more biomedcentral.com/submissions", "section_num": null } ]	[ { "section_content": "We would like to thank Dr. Jinwen Zhang for giving instruction in the bioinformatic analysis. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "This work was supported by grants from the National Natural Science Foundation of China (Grant # 82170185, 81720108002, 81970146, 81770166), Natural Science Foundation of Jiangsu Province (BK20211376), Translational research grant of NCRCH (2020ZKZB01), CSCO Research Foundation (Y-Roche2019/2-0090) and Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University (PY2021036). ", "section_name": "Funding", "section_num": null }, { "section_content": "The datasets generated and/or analysed during the current study are available in the National Center for Biotechnology Information Sequence Read Archive (SRA), PRJNA762572. ", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s40164-022-00302-0. Additional file 2: Table S2.Metabolic score of 53 CLL patients Additional file 3: Table S3.Genes that positively correlated with cir-cRIC8B (R>0.5).S4.Differentially expressed metabolites analyzed by positive ion mode. Author contributions HJ, JL, LF and ZW designed the research and supervised the project.ZW, DG, RW, XZ, LW and XL executed all experiments.HZ, SQ, YX and WZ performed statistical analysis of data.HZ and WX was responsible for clinical sample collection.ZW and DG wrote the manuscript.All authors read and approved the final manuscript. The study was authorized by the Ethics Committee of Jiangsu Province Hospital.All patients signed consent forms. Not applicable. The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Supplementary Information", "section_num": null }, { "section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s40164-022-00302-0. ", "section_name": "Supplementary Information", "section_num": null }, { "section_content": "Additional file 2: Table S2.Metabolic score of 53 CLL patients Additional file 3: Table S3.Genes that positively correlated with cir-cRIC8B (R>0.5).S4.Differentially expressed metabolites analyzed by positive ion mode. ", "section_name": "Additional file 1: Table S1. Primer sequences used in qRT-PCR and PCR analysis", "section_num": null }, { "section_content": "Author contributions HJ, JL, LF and ZW designed the research and supervised the project.ZW, DG, RW, XZ, LW and XL executed all experiments.HZ, SQ, YX and WZ performed statistical analysis of data.HZ and WX was responsible for clinical sample collection.ZW and DG wrote the manuscript.All authors read and approved the final manuscript. ", "section_name": "Additional file 4:", "section_num": null }, { "section_content": "The study was authorized by the Ethics Committee of Jiangsu Province Hospital.All patients signed consent forms. ", "section_name": "Declarations Ethics approval and consent to participate", "section_num": null }, { "section_content": "Not applicable. ", "section_name": "Consent for publication", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.1186/s12979-019-0163-x	The presence of CLL-associated stereotypic B cell receptors in the normal BCR repertoire from healthy individuals increases with age	Aging is known to induce immunosenescence, resulting in alterations in both the innate and adaptive immune system. Here we evaluated the effects of aging on B cell subsets in peripheral blood of 155 immunologically healthy individuals in four age categories (range 20-95y) via multi-parameter flow cytometry. Furthermore, we studied the naive and antigen-experienced B cell receptor (BCR) repertoire of different age groups and compared it to the clonal BCR repertoire of chronic lymphocytic leukemia (CLL), a disease typically presenting in elderly individuals.Total numbers and relative frequencies of B cells were found to decline upon aging, with reductions in transitional B cells, memory cell types, and plasma blasts in the 70 + y group. The BCR repertoire of naive mature B cells and antigen-experienced B cells did not clearly alter until age 70y. Clear changes in IGHV gene usage were observed in naive mature B cells of 70 + y individuals, with a transitional pattern in the 50-70y group. IGHV gene usage of naive mature B cells of the 50-70y, but not the 70 + y, age group resembled that of both younger (50-70y) and older (70 + y) CLL patients. Additionally, CLL-associated stereotypic BCR were found as part of the healthy control BCR repertoire, with an age-associated increase in frequency of several stereotypic BCR (particularly subsets #2 and #5).Composition of the peripheral B cell compartment changes with ageing, with clear reductions in non-switched and CD27 + IgG+ switched memory B cells and plasma blasts in especially the 70 + y group. The BCR repertoire is relatively stable until 70y, whereafter differences in IGHV gene usage are seen. Upon ageing, an increasing trend in the occurrence of particular CLL-associated stereotypic BCR is observed.	[ { "section_content": "Changes in the immune system related to aging generally lead to increased susceptibility to infections, poor responses to new and evolving pathogens, poor vaccination responses, and higher incidence of autoimmune disorders and malignancies [1,2].This decline in function of the immune system, also referred to as immunosenescence, is the result of alterations occurring in both innate and adaptive immunity [3]. Age-related changes in humoral immune responses have generally been ascribed to defects in the T cell compartment and a lack of T cell help for B cell function [3].Nevertheless, mouse studies do provide evidence for changes in the B cell compartment itself during aging.Although total B cell numbers did not alter much, shifts in the distribution of functional subsets were apparent with old age.In fact, in old mice nearly 100% of splenic B cells exhibited an antigen-experienced phenotype [4] and circulating immunoglobulins (Ig) were predominantly derived from post-germinal center B cells, as deduced from the presence of somatic hyper mutations (SHM) [5]. In human, age-related alterations in peripheral blood (PB) B cell subset distribution have also been reported, with circulating CD19+ B cells declining in absolute numbers and frequencies [6][7][8][9][10].In some studies numbers and percentages of CD27+ memory B cells were found to decline [7,8], whereas others showed an increase of these cells [10][11][12].Similarly, numbers and percentages of naive CD27-IgD+ B cells were found to decrease by some studies [9,10,12], whereas others reported an increase [7,8].These inconsistent results may be explained by different B cell subset definitions and/or by large inter-individual variations in the studied age groups [11,13]. Changes in B cell subsets during aging will likely also impact on B cell receptor (BCR) repertoire diversity.Indeed, in several mouse models age-related changes in the naive BCR repertoire were reported [14].In some elderly humans, Ig heavy chain (IGH) complementarity determining region 3 (HCDR3) spectratyping analysis of PB B cells revealed a significant loss of diversity, which was associated with poor health status and poor survival [13].Conflicting data, however, were reported on SHM in IGHV genes of the memory B cell compartment upon aging, varying from increased mutation rates in IgG+ but not in IgM+ PB memory B cells, to increased mutation rates in IgM+ memory B cells but not in other tonsillar subsets [15,16]. Introduction of next generation sequencing (NGS) technologies has opened new possibilities to analyze the aging BCR repertoire, particularly in the light of immune diseases that typically arise in elderly.One category of immune diseases with a higher change to develop in elderly humans are B cell malignancies, with chronic lymphocytic leukemia (CLL) being the most common type.Notably, in about one-third of CLL patients, quasiidentical (stereotypic) BCRs are observed, which are characterized by restricted IGHV, IGHD, and IGHJ gene usage plus similarities in HCDR3 length and amino acid sequence [17].One study reported on stereotypic BCRs within the normal IGHV1-69-IGHJ6 repertoire [18].Little is known however about the overall existence of CLL-associated stereotypic BCRs in the normal BCR repertoire of different age groups.We hypothesize that these CLL-associated stereotypic BCR could be a reflection of changes in B cell subset distribution and the normal BCR repertoire upon aging. In this study we determined absolute numbers and relative distribution of PB B cell subsets in healthy individuals of different age categories.Additionally, we used NGS to investigate the BCR repertoire of naive mature B cells and different types of antigen-experienced B cells in healthy individuals upon aging.Finally, we compared IGHV gene usage of the normal BCR repertoire of different age groups with that of CLL leukemic cells and evaluated the occurrence of CLL-associated stereotypic BCR in the aging normal BCR repertoire.To validate our cohort, we first evaluated age-related dynamics of the total white blood cells and lymphocyte subpopulations (Fig. 1; Additional file 1: Figure 1).White blood counts (WBC) remained stable across the age groups (Fig. 1A).Although differences were not significant, there was a trend that the absolute numbers of lymphocytes was lower in the 50-60y and 60-70y groups than in the <50y group (Additional file 1: Figure S1).Both absolute and relative numbers of naive CD8+ T cells significantly declined >50y of age, whereas absolute and relative numbers of CD8+ effector (TemRA) T cells clearly increased with increasing age and CD8+ effector memory (TemRO) T cells remained stable (data not shown).CD4+ T cell and NK cell numbers did not alter between the age groups (Additional file 1: Figure S1).These data are in line with previously reported data on T and NK cells [19], thus supporting the validity of our cohort for evaluating B cell aging effects. ", "section_name": "Background", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Next, we focused on the composition of the B cell compartment (see Methods, Additional file 6: Table S1, Additional file 2: Figure S2) in the different age groups.The total B cell numbers and relative frequencies of B cells (as fraction of total lymphocytes) slightly declined during aging, resulting in a significant difference between the <50y and 70 + y groups (Fig. 1B-C).More specifically, we observed a significant reduction in absolute numbers of transitional B cells in the 70 + y group, as well as reductions in the non-switched and CD27 + IgG+ switched memory B cell populations, and plasma blasts in the two oldest age categories (Fig. 1D-M).For naive mature B cells and all other types of memory B cells no clear alterations in absolute numbers were noted upon aging (Fig. 1D-M).The overall PB B cell subset distribution displayed only minor shifts between different age groups (Fig. 1N), which was mainly reflected by the significantly lower frequencies of non-switched memory B cells and plasma blasts upon increasing age (Additional file 3: Figure S3). We then looked into CD5 + CD43+ chronically activated B cells, as these have been associated with CLL and CLL-like MBL that typically appear in elderly [20][21][22].We noted a small but significant increase in relative frequencies of CD5 + CD43+ B cells in the 70 + y group (when compared with the 60-70y age group), together with a trend towards increased absolute numbers of these cells (Fig. 2A-B).Next, we also evaluated CD21 low B cells, as high numbers of these cells have been associated with autoimmune disease [23].Notably, a significant increase of CD21 low cell numbers and relative frequencies was seen between the 50-60y and 60-70y groups, which normalized again in the 70 + y group (Figs.2C-D). Taken together, our B cell subpopulation analysis mostly showed a decline in transitional B cells, non-switched and CD27 + IgG+ switched memory B cells, and plasma blasts in elderly.The frequency of CD21 low B cells appeared to be increased in 60-70y group. Composition of the BCR repertoire of naïve mature B cells is stable until 70y but shows changes in the 70 + y group Our next aim was to see which differences occur in the BCR repertoire of healthy donors during aging.To this end, we first sorted antigen-inexperienced naive mature B cells of healthy controls in the <50y, 50-70y, and 70 + y groups (n = 4-5 per age group) and analyzed unique IGHV-IGHD-IGHJ sequences and their HCDR3 regions (Additional file 6: Table S2).Despite slight variations in the mean HCDR3 lengths between the <50y (53.0 nucleotides), 50-70y (54.5 nucleotides), and 70 + y (46.1 nucleotides) groups, the overall HCDR3 profiles showed no significant differences (Fig. 3A). When evaluating gene usage, differences in IGHV subgroup usage between the <50y and 50-70y groups appeared limited, but we did find a marked increase in IGHV1 and IGHV5 subgroup usage and a decrease in IGHV4 subgroup usage in the 70 + y donors (Fig. 3B).Upon further examination of IGHV gene usage, significant differences were mostly noted in the 70 + y age group, with IGHV1-18, IGHV1-46, IGHV1-69, and IGHV5-51 gene usage being significantly higher, and IGHV4-34, IGHV4-39, and IGHV4-59 usage being significantly lower (Fig. 3C), which could not be explained by small clonal proliferations.That said, the most commonly used IGHV gene in all three age groups appeared to be the IGHV3-23 gene, followed by IGHV3-21 (Fig. 3B).We did not detect clear differences in IGHD and IGHJ gene usage between any of the three age groups. Collectively, our data from healthy controls of different age groups suggest that the BCR repertoire of naive mature B cells is relatively stable until 70y.In contrast, in the 70 + y group IGHV gene usage does differ, which most probably should be interpreted as an aging effect, since there were no other indications that can explain this difference. ", "section_name": "Alterations", "section_num": null }, { "section_content": "To evaluate age effects in the antigen-selected BCR repertoire, we then focused on non-switched, IgM-only, and CD27 + IgG+ switched memory B cells, which are all antigen-experienced cells though arising via distinct activation routes.Some of the memory B cell populations are so small that they can only be sorted from buffy coats of healthy donors (n = 4-5 per age group); unfortunately blood donors are only allowed to give blood until 70y, so we could not the study the 70 + y age group.Mean HCDR3 lengths did not differ significantly between the <50y and 50-70y age groups (Additional file 4: Figure S4).IGHV, IGHD, and IGHJ gene usage and combined IGHV / IGHJ patterns of nonswitched, IgM-only, and CD27 + IgG+ memory B cells did not show significant differences either between these two age groups (Additional file 5: Figure S5).Notably, when analyzing SHM frequencies for these memory B cell subpopulations, we did detect a higher mutation rate for non-switched and IgM-only memory B cells in the 50-70y group, whilst a small reduction in mutation frequency was seen in CD27 + IgG+ switched memory B cells (Additional file 5: Figure S5). Collectively these data show that the BCR characteristics of memory B cell subpopulations do not differ statistically between the <50y and 50-70y groups.IGHV gene usage in the clonal BCR repertoire of CLL patients is largely comparable to IGHV genes in naive mature B cells of 50-70y individuals As the BCR is known to play an important role in disease onset and prognosis of CLL, which normally develops at elderly age (average 70 + y), we then asked whether overall IGHV gene usage in CLL patients of different age groups would reflect the BCR repertoire of normal B cells of the same age groups.To this end we evaluated IGHV gene usage in different B cell subsets of healthy controls of different age groups and compared the profiles with Sanger sequencing-based data of a cohort of 920 CLL patients (Fig. 4). Overall IGHV gene usage profiles did not differ significantly between different types of normal B cell subpopulations, such as naive mature B cells, nonswitched memory B cells, and IgM-only memory B cells, in the 50-70y age group (p = 0.99-1 in all comparisons).Unfortunately, no such comparison was possible in 70 + y individuals, due to the lack of available data from non-switched and IgM-only memory B cells for this age group.However, overall IGHV gene usage profiles in naive mature B cells of healthy individuals did clearly vary between the 50-70y and 70 + y groups (p = 0.005). When we next compared overall IGHV gene usage profiles between CLL patients of the 50-70y and the 70 + y groups we did not observe a significant difference (p = 0.995).The IGHV profiles of naive mature B cells and CLLs in the 50-70y group appeared to look rather similar (p = 0.574), albeit with a more dominant IGHV1-69 and IGHV4-34 usage in CLL.In contrast, naive mature B cell and CLL IGHV gene usage profiles in the 70 + y groups were clearly different (p < 0.0001), while the overall IGHV profile in 70 + y CLL was in fact rather similar to that of naive mature B cells of the 50-70y age group (p = 0.110).Furthermore, overall IGHV profiles in the 50-70y group appeared clearly different between CLL and non-switched memory B cells (p = 0.028) or IgM-only memory B cells (p = 0.004). These data indicate that the overall IGHV gene usage profile in CLL patients, irrespective of the age of presentation, is similar to naive mature B cells of especially the 50-70y control group.The overall IGHV profile of the 70 + y control group is different without obvious explanation as mentioned above. ", "section_name": "Differences in BCR repertoire of memory B cell subpopulations are minor between <50y and 50-70y age groups", "section_num": null }, { "section_content": "In view of the occurrence of quasi-identical (stereotypic) BCR with similar IGHV / IGHD / IGHJ and HCDR3 features in CLL clones of different patients, we then investigated whether we could also detect stereotypic BCR in healthy individuals of different age groups.To identify CLL-related stereotypic BCRs in the normal repertoire of healthy controls of different age groups, a reference database of stereotypic BCR from CLL patients [24] was used for assigning individual IGH sequences from healthy controls.With this algorithm we could indeed identify CLLassociated stereotypic BCRs based on HCDR3 characteristics in the repertoire of naive mature B cells, and also of non-switched, IgM-only, and CD27 + IgG+ switched memory B cells (Fig. 5).The presence of stereotypic BCR receptors in naive mature B cells showed an increasing trend with age and was most apparent in the 70 + y group (Fig. 5A).In naive mature B cells, the most prominent stereotypic BCR belonged to CLL subsets #2, #5, and #64B (Fig. 5B).In non-switched memory B cells the most frequently found stereotypic BCRs concerned CLL subsets #2 and #14 (Fig. 5C).The CLL#14 BCR also appeared most prominent in IgM-only and CD27 + IgG+ switched memory B cells, especially in the 50-70y category (Fig. 5D-E). From these findings we conclude that CLL-associated stereotypic BCRs are present in the normal BCR repertoire during life and that their frequency showed an increasing trend upon aging. ", "section_name": "CLL-associated stereotypic BCR are present in naive mature B cells and increase with age", "section_num": null }, { "section_content": "Here we evaluated age-related changes in B cell subset composition as well as BCR repertoire with a focus on CLL-associated stereotypic BCR usage.In the most elderly (70 + y) individuals we observed a decline in absolute numbers of transitional B cells, total memory B cells, and plasma blasts.The BCR repertoire of naive mature B cells and distinct memory B cell populations was found to be relatively stable until 70y.In naive mature B cells of the 70 + y group differences were noted, especially with respect to IGHV gene usage.Notably, IGHV gene usage in the clonal BCR repertoire in CLL did not differ with the age of presentation of the CLL patients, and largely resembled naive mature B cells of the 50-70y group rather than the 70 + y group.Finally, CLL-associated stereotypic BCR were found as part of The observed decline in total B cell numbers in the 70 + y group could result from a reduced output from the bone marrow [25,26].An alternative explanation could be the reduction in CD27+ antigen-experienced B cell subpopulations.Non-switched and CD27 + IgG+ memory B cells together make up the majority of the CD27+ memory B cell compartment, and were previously described to decline upon aging [6].The decline in non-switched memory B cells could underlie the higher vulnerability to diseases caused by encapsulated bacteria like Streptococcus pneumoniae in elderly [27].CD27 + IgG+ memory B cells are mainly, albeit not exclusively, formed in T cell-dependent immune responses and play a role in recall responses to previously encountered pathogens [28].The reduction of plasma blasts upon aging is in line with earlier observations [29] and fits the lower immunoglobulin levels in the circulation as reported in elderly [30].Together these data could, at least partially, explain the reduced effects of vaccination and immune responses against infections in elderly. Chronically activated B cells express CD5 and CD43 [31,32] and might trigger MBL onset.[33,34].MBL are found in healthy adult individuals, with an incidence that increases with age to roughly 10% of individuals >65y [35].Based on their phenotypical association with MBL [20] and CLL [22], the increase of CD5 + CD43+ B cells upon aging might thus correlate with the higher risk of MBL and CLL clones in elderly.Another B cell subset related with chronic activation concerns CD21 low B cells, increased numbers of which can be found in patients showing chronic inflammation in the context of autoimmune disease [23].As we excluded individuals with inflammatory and (auto)immune disease in our immunologically healthy cohort, unfortunately we could not link the higher number of CD21 low B cells in the 60-70y group to overt autoimmune disease occurrence.Nevertheless, increased numbers of CD21 low B cells in this age group might reflect an BCR repertoire changes were most apparent in naive mature B cells of the 70 + y group.Since naive mature B cells are not affected by exogenous antigen, this is most likely the result of changes in repertoire development and/ or output from the bone marrow.Whilst HCDR3 length, IGHD, and IGHJ usage remained stable in all three age categories, IGHV gene usage did reveal alterations.Interestingly, IGHV4-34 usage, a gene often associated with autoimmunity, was found to be reduced upon aging in these healthy individuals.Upon aging we also noticed a combined increased usage of IGHV5-51 and IGHV1-69, in line with a previous report [36].IGHV1-69 has been associated with broadly neutralizing antibodies against amongst others influenza, HIV, hepatitis C, and commensal bacteria antigens in the context of CLL [37]. Remarkably, IGHV gene usage in both the 50-70y and 70 + y CLL patient groups most closely resembled IGHV gene usage in naive mature B cells of 50-70y, but not 70 + y, healthy individuals.One could speculate that CLL clones, even the ones in elderly CLL patients (70 + y), would have developed from B cells with a BCR repertoire of relatively younger age (<70y), but it might also reflect selection for IGHV specificities in the younger repertoire that could be predisposing for CLL development. Stereotypic BCR, which are found in roughly one third of CLL clones, have previously been documented in RNA from a total lymphocyte pool of three healthy individuals (age 50, 69, and 69) [38,39].In this study we show that stereotypic BCR can be found in the normal BCR repertoire of both naive mature and antigen-experienced B cells.Interestingly, a frequently observed stereotypic BCR was the CLL#2 BCR (IGHV3-21 in combination with IGHJ6 with a short HCDR3 length of 9 amino acids), which is the most common stereotypic BCR seen in CLL patients and is associated with an aggressive form of CLL [17,40].Stereotypic CLL#5 BCR (IGHV1-69, IGHD3-10/3-3, IGHJ6, 20 amino acids HCDR3) as well as stereotypic CLL#64 BCR (IGHV3 subgroup genes, IGHD2 subgroup genes, IGHJ6, 21 amino acids HCDR3) [17] were relatively often detected in naive mature B cells of especially individuals 70 + y.In antigen-experienced B cells, stereotypic CLL#14 BCR (IGHV4-4, no specific IGHD, IGHJ4, and short 10 amino acids HCDR3) was frequently found.The possibility to detect stereotypic BCRs with short HCDR3 lengths in antigen-experienced B cell subpopulations would be in line with our observation that on average the complete memory BCR repertoire shows selection for shorter HCDR3 lengths in comparison with naive B cells.Notably, other common CLL-associated stereotypic BCR, such as CLL#4, CLL#6, and CLL#8 BCR, could not be detected. Even though the frequency of stereotypic BCR in healthy B cells shows a trend towards increase with age, stereotypic BCR can already be detected in cord blood (data not shown) and thus are to be considered as part of the normal BCR repertoire.Moreover, even though the increase of CLL-associated stereotypic BCR in the aging normal BCR repertoire might imply an increased predisposition for CLL development in elderly, it should be stressed that two thirds of CLL show heterogeneous BCR specificities that can also mediate derailment of B cells leading to CLL.Investigations into the presence of CLLassociated stereotypic BCR in healthy individuals should therefore be extended to larger datasets including more healthy individuals of all age groups, as well as to individuals suffering from chronic infection, immunodeficiency, or autoimmune disease.Such studies would allow to define the true impact of CLL-associated stereotypic BCR in CLL development. ", "section_name": "Discussion", "section_num": null }, { "section_content": "We analyzed the peripheral B cell compartment and BCR repertoire during human ageing.Composition of the peripheral B cell compartment changes with ageing, with clear reductions in non-switched and CD27 + IgG+ switched memory B cells and plasma blasts in especially the 70 + y group.The BCR repertoire is relatively stable until 70y age, whereafter differences in IGHV gene usage are seen.Upon ageing, an increase in the occurrence of particular CLL-associated stereotypic BCR is observed, potentially reflecting the occurrence of such BCR in CLL in elderly. ", "section_name": "Conclusion", "section_num": null }, { "section_content": "", "section_name": "Methods", "section_num": null }, { "section_content": "For B cell subpopulation analysis, peripheral blood (PB) of immunologically healthy individuals was obtained from pre-surgery patients (Dept.Orthopedics, Erasmus MC) with the following exclusion criteria: (auto)immune or inflammatory diseases; malignancies; usage of anti-inflammatory or immunosuppressive drugs; surgery in the past 30 days; alcohol and drug abuse.To increase the number of subjects per age group, additional samples from the SENEX healthy aging cohort (Rheumatology and Clinical Immunology, UMCG, Groningen, Netherlands), and samples from co-workers from the department were included.Subjects (n = 155) were divided into four age categories: < 50 (n = 47), 50-60 (n = 31), 60-70 (n = 45), and 70+ (n = 32).For the BCR repertoire study, peripheral blood samples (n = 5 per age group) were additionally obtained from Sanquin Blood bank (Amsterdam, The Netherlands).Diagnostic samples from CLL patients were collected upon informed consent and anonymized for further usage.Written consent was obtained in accordance with the Declaration of Helsinki after medical ethics committee approval (MEC 2011-409, 2016-202, and 2,012,375). ", "section_name": "Sample inclusion", "section_num": null }, { "section_content": "Folllowing white blood cell count (WBC) measurement on a Coulter®Ac.T diff analyzer (Beckman Coulter, Fullerton, CA, USA), flowcytometry was performed on whole blood [after red blood cell lysis with ammonium chloride] using an LSR Fortessa™ (BD Biosciences, San Jose, CA).Absolute cell counts of monocytes, natural killer (NK) cells, T cells, and B cells were calculated from WBC numbers using Infinicyt software (Cytognos, Salamanca, Spain).Lymphocytes were first gated based on FSC / SSC characteristics, and B cells were defined by expression of the pan-B cell marker CD19.Further gating was performed for defined CD19+ B cell subpopulations, i.e. transitional B cells (CD38hi/CD27-), naive mature B cells (CD38-/CD27-/IgM+/IgD+), non-switched memory B cells (CD38-/CD27+/IgM+/IgD+), IgM-only B cells (CD38-/CD27+/IgM+/IgD-), switched memory B cells (CD38-/CD27+ or -/IgM-/IgD-IgG+ or IgA+ or IgE+), plasma blasts (CD38 hi /CD27+), CD5+/CD43+ B cells (CD38-/CD5+/CD43+), and CD21 low B cells (CD38 dim / CD21 low ) according to published data [28] (see also (Additional file 6: Table S1), using 11-color flowcytometric stainings (Additional file 6: Table S3) and FACS DIVA software (BD Biosciences) for analysis. ", "section_name": "Immunophenotyping of B cell subpopulations", "section_num": null }, { "section_content": "PB mononuclear cells (PB-MNC) were isolated via Ficoll Paque gradient centrifugation.Subsequently, B cells were purified with human CD19 MicroBeads via AutoMACS (Myltenyi Biotech, Bergisch Gladbach, Germany).Next, several B cell subpopulations (naive mature, non-switched memory, IgM only, and CD27+/IgG+) were collected using a FACSAria cell sorter (BD Biosciences).Immediately after collection cells were lysed in RLT+ buffer (QIAGEN, Valencia, CA) complemented with β-mercapto-ethanol.Cells were used for DNA isolation with the DNA/RNA/ miRNA Easy kit (QIAGEN) and/or stored in -80 °C for later processing. ", "section_name": "Sorting of B cell subpopulations and DNA isolation", "section_num": null }, { "section_content": "IGHV-IGHD-IGHJ rearrangements were amplified from 100 ng DNA of sorted B cell subpopulations (from n = 3-5 healthy controls per age group; see Additional file 6: Table S3) using the BIOMED-2 IGH multiplex PCR with 6 IGHV primers and 1 IGHJ consensus primers that were extended with adapter sequences for NGS.PCR products were purified by gel extraction (QIAGEN) and subsequently by Agencourt AMpure XP beads (Beckman Coulter, Brea, CA).Concentrations were measured using Quant-iT Picogreen dsDNA assay (Invitrogen, Carlsbad, CA).PCR products were sequenced on a 454 GS junior (Roche, Branford, CT), using the GS Junior Titanium emPCR, sequencing, and PicoTiterPlate kits (Roche), and partly on a MiSeq (Illumina, San Diego, CA) platform.Cross-validation experiments using B cells from healthy individuals showed comparability of data from both platforms (unpublished; see Additional file 7).Sequences were demultiplexed based on their multiplex identifier sequence and trimmed via the ImmunoGlobulin galaxy (IGGalaxy) pipeline [41].FASTA files were uploaded in IMGT/High-V-QUEST (http://www.imgt.org/HighV-QUEST/login.action) and IMGT output files were further analyzed in the Antigen Receptor Galaxy pipeline, as described before [42]. For comparison purposes, a local Erasmus MC cohort of 920 CLL patients (mean 65y) was used, in which Sanger sequencing-based IGHV mutation status analysis was performed using the BIOMED-2 primers and protocol [43], and following ERIC interpretation guidelines [44]. IGHV-IGHJ circos plots were generated via the Circos Table Viewer (http://mkweb.bcgsc.ca/tableviewer/).CLLassociated stereotypic BCR in healthy control samples were defined using the ARResT/AssignSubsets tool (http://tools.bat.infspire.org/arrest/assignsubsets/). ", "section_name": "NGS-based BCR repertoire analysis of healthy individuals", "section_num": null }, { "section_content": "Significant differences in relative and absolute numbers of lymphocyte subpopulations and IGH SHM levels between age groups were determined using the Mann-Whitney U test.Differences in HCDR3 lengths were evaluated using Kolmogorov-Smirnov statistics.Relative IGHV gene usage and relative frequencies of stereotyped BCR between different age groups were analyzed using a two-way ANOVA with multiple comparisons.Overall IGHV gene usage between B cell subpopulations and CLL clones was analyzed using the Fisher's exact test.A p-value < 0.05 was considered significant.Statistics were performed in GraphPad Prism v5.0 (La Jolla, CA). ", "section_name": "Statistical analysis", "section_num": null } ]	[ { "section_content": "The authors wish to thank Michèle van der Klift, Irene van der Linden, Kim Heezen, Tamara Wabeke, Joyce Schilperoord-Vermeulen, Ellen van Gastel-Mol, Diana van den Heuvel, and Pauline van Schouwenburg for excellent technical and analytical support.The research for this manuscript was performed within the framework of the Erasmus MC Postgraduate School Molecular Medicine. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "This study was financially supported through an unrestricted grant from Roche-Genentech (to AWL and JJMvD), and a Ministry of Health of the Czech Republic grant 16-34272A (to ND). ", "section_name": "Funding", "section_num": null }, { "section_content": "The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. ", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "Additional file 1: Ethical approval and written consent to participate was obtained from the medical ethics committee of Erasmus MC, Rotterdam (MEC 2011-409, 2016-202) and UMCG, Groningen (2012375). Written consent for publication was obtained from the medical ethics committee of Erasmus MC, Rotterdam (MEC 2011-409, 2016-202) and UMCG, Groningen (2012375). The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Additional files", "section_num": null }, { "section_content": "Additional file 1: ", "section_name": "Additional files", "section_num": null }, { "section_content": "Ethical approval and written consent to participate was obtained from the medical ethics committee of Erasmus MC, Rotterdam (MEC 2011-409, 2016-202) and UMCG, Groningen (2012375). ", "section_name": "Ethics approval and consent to participate", "section_num": null }, { "section_content": "Written consent for publication was obtained from the medical ethics committee of Erasmus MC, Rotterdam (MEC 2011-409, 2016-202) and UMCG, Groningen (2012375). ", "section_name": "Consent for publication", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.1007/s44228-022-00020-8	Effectiveness and Safety of Ibrutinib for Chronic Lymphocytic Leukemia in Routine Clinical Practice: 3-Year Follow-up of the Belgian Ibrutinib Real-World Data (BiRD) Study	<jats:title>Abstract</jats:title><jats:p>The multicenter observational BiRD study investigated the real-world effectiveness and safety of ibrutinib in patients with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and Waldenström’s macroglobulinemia (WM) in Belgium. This interim analysis reports results for patients with CLL, with a median follow-up of 34 months. Overall, patients had predominantly relapsed/refractory disease (73%) and were elderly (median age 72 years) with high-risk features such as del17p and/or <jats:italic>TP53</jats:italic> mutations (59%). Patients were included either prospectively or retrospectively, and the total patient population effectiveness results were adjusted with left truncation. In the effectiveness population (<jats:italic>N</jats:italic> = 221: prospective, <jats:italic>n</jats:italic> = 71; retrospective, <jats:italic>n</jats:italic> = 150), the overall response rate was 90.0%. Median progression-free survival was 38.3 months (prospective, not estimable; retrospective, 51.5 months) and median overall survival was not yet estimable in the total, prospective and retrospective groups. Treatment-emergent adverse events (TEAEs) for the prospective and retrospective groups are reported separately. Any-grade TEAEs of interest in the prospective/retrospective groups included infections (67.1%/60.1%), diarrhea (20.5%/10.5%), hypertension (16.4%/9.8%) and atrial fibrillation (12.3%/7.2%). Major bleeding was reported in 5.5%/3.3% of prospective/retrospective patients, with little difference observed between those receiving versus not receiving antithrombotic treatment. Discontinuations due to toxicity were reported in 10.5% of patients. Results from this interim analysis show treatment with ibrutinib to be effective and tolerable, with no new safety signals observed. Future analyses will report on longer-term follow-up. </jats:p>	[ { "section_content": "Ibrutinib is a first-in-class, once-daily oral inhibitor of Bruton's tyrosine kinase (BTK) approved in Europe as monotherapy for the treatment of adult patients with previously untreated and relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) [1]. Ibrutinib has demonstrated efficacy in treating patients with CLL in a number of phase 3 trials.Ibrutinib monotherapy showed improved progression-free survival (PFS) and overall survival (OS) versus chlorambucil in previously untreated patients with CLL aged ≥ 65 years (RESO-NATE-2™) [2], and also versus ofatumumab in patients with R/R CLL (RESONATE™) [3].When used as a single agent or in combination with rituximab, ibrutinib therapy showed improved PFS compared with bendamustine plus rituximab in previously untreated patients with CLL aged ≥ 65 years (Alliance) [4].It also improved PFS and OS as part of combination therapy with rituximab versus fludarabine, cyclophosphamide and rituximab (FCR) in previously untreated patients aged ≤ 70 years (E1912) [5].Improved PFS was also shown for ibrutinib in combination with obinutuzumab versus chlorambucil plus obinutuzumab in previously untreated CLL (iLLUMINATE) [6] and in combination with bendamustine and rituximab (BR) versus placebo plus BR in patients with R/R CLL (HELIOS) [7,8]. The Belgian ibrutinib Real-World Data study (BiRD) was designed to investigate the effectiveness and safety of ibrutinib treatment in real-world clinical practice in patients with CLL, MCL or Waldenström's macroglobulinemia (WM) in Belgium.Results from first and second interim analyses from BiRD have been presented previously in patients with CLL and MCL [9][10][11], and some of the third interim analysis results in patients with CLL and MCL have also been presented [12].Here we report the results for the cohort of patients with CLL from the third interim analysis of the BiRD study. ", "section_name": "Introduction", "section_num": "1" }, { "section_content": "", "section_name": "Patients and Methods", "section_num": "2" }, { "section_content": "BiRD is a retrospective and prospective, multicenter, noninterventional observational study of adult patients with a confirmed diagnosis of CLL, MCL or WM from 26 sites in Belgium who initiated reimbursed ibrutinib therapy on or after its commercial availability in Belgium (August 1, 2015 for R/R CLL and MCL, September 1, 2016 for WM and May 1, 2017 for first-line CLL), or who participated in the Medical Need Program for CLL, WM or MCL, or received free of charge ibrutinib as a single-patient request for WM and switched to reimbursed ibrutinib.Patients who initiated reimbursed ibrutinib therapy prior to the inclusion visit were eligible for retrospective enrollment in the study, regardless of whether the ibrutinib therapy was ongoing at the time of inclusion (retrospective inclusion).Patients who had begun ibrutinib therapy at the time of inclusion in the study were added prospectively.This is the third of four planned interim analyses.The first occurred after the last patient was enrolled, the second after 18 months, this present and third at 3 years and the fourth is planned for early 2023.However, ad hoc interim analyses may also be conducted by the sponsor, based on specific questions from the medical field or from health authorities.Only patients with CLL are included in this analysis, and the cutoff date was March 31, 2020. Each patient was treated according to the standard of care; their decision to take part in the observational study did not influence their medical care.Therapy decisions were made at the discretion of the participating physician, according to routine clinical practice. Data were collected every 3 months during the first year and every 6 months thereafter for a period of 5 years.Data were collected for ibrutinib treatment until discontinuation, after which the follow-up period for response started with any subsequent therapy.If patients received no further treatment, they were followed up for survival only.Safety data 1 3 were collected for some patients who began ibrutinib before inclusion and yet continued with ibrutinib therapy; therefore, there was a retrospective/prospective safety data collection period for these patients-defined within the retrospective group as \"before inclusion\" and \"after inclusion.\" The study protocol was approved by the institutional review boards or independent ethics committees of participating centers.All patients received oral and written information on the study and provided informed consent to data collection and source data verification. ", "section_name": "Study Design", "section_num": "2.1" }, { "section_content": "Patients had to be aged ≥ 18 years, with a confirmed diagnosis of CLL, and eligible for reimbursed ibrutinib treatment, according to the National Institute for Health and Disability Insurance (RIZIV/INAMI).This was both before and after the commercial availability of ibrutinib and, therefore, also included patients participating in a Medical Need Program who were switched to reimbursed ibrutinib treatment when it became available (August 1, 2015).Previously untreated patients without del17p or TP53 mutation who were fit to receive fludarabine-based regimens were not eligible for ibrutinib treatment according to Belgian treatment guidelines at the start of the study.Reimbursement criteria for CLL are based on the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) criteria [13] and include del17p or TP53 mutation or R/R disease.Patients were excluded if they were participating in another investigational or clinical study or in any expanded access program during the ibrutinib treatment period covered by this observational study. ", "section_name": "Inclusion and Exclusion Criteria", "section_num": "2.2" }, { "section_content": "The primary outcome measures were investigator-assessed overall response rate (ORR) and PFS.Secondary outcome measures included OS and safety, which includes treatment exposure and treatment-emergent adverse events (TEAEs). PFS was defined as the time from the start of ibrutinib treatment to progression or death from any cause.OS was defined as the time in months from initiation of ibrutinib to death.ORR was the sum of complete response (CR), partial response (PR) and partial response with lymphocytosis, as assessed by the investigator.Although confirmation of CR by minimal residual disease analysis of bone marrow was possible, it is not routinely performed in real-world practice, and this definition of CR should be considered unconfirmed in most cases.After discontinuation of ibrutinib therapy, during the treatment-free period, patients were followed for response or disease progression every 6 months ± 2 weeks.If no visit was conducted within this recommended period, data from the visit conducted as close as possible to the protocol-specified time point were recorded. All TEAEs were collected prospectively, whereas only adverse drug reactions considered related to ibrutinib were collected retrospectively. ", "section_name": "Outcome Measures", "section_num": "2.3" }, { "section_content": "This study describes everyday ibrutinib treatment practice for CLL.The effectiveness population included all patients who met the inclusion criteria and who received at least one dose of ibrutinib and the safety population included all patients who received at least one dose of ibrutinib. All time-to-event variables were analyzed using standard survival analysis methods, including Kaplan-Meier product-limit survival curve.The median time to event with two-sided 95% confidence intervals (CIs) was estimated.All continuous variables were summarized using descriptive statistics, which included the number of patients, mean, standard deviation, median, interquartile range (IQR) minimum and maximum and 95% CI.Data were reported for the prospective, retrospective and/or total patient populations for the effectiveness population.Owing to the differences in adverse event collection between the retrospective and prospective groups, these data are presented separately and are all referred to as \"TEAEs\" for ease of notation.The number and percentage of patients who experienced at least one occurrence of each TEAE (presented as preferred term and categorized by system organ class) were summarized.TEAEs were not graded but were summarized by severity for serious and severe TEAEs, according to the worst experienced.Patients who died before enrollment in the trial could not be included according to Belgian law, as no informed consent could be obtained.For this reason, the total population effectiveness results were adjusted with left truncation, which reduces part of the follow-up time of the retrospective patients to avoid any mortality bias.However, this shortened the median time to event and, as the retrospective group is larger than the prospective group, this also affected the median time to event of the combined analysis. ", "section_name": "Statistics", "section_num": "2.4" }, { "section_content": "", "section_name": "Results", "section_num": "3" }, { "section_content": "A total of 221 evaluable patients with CLL were included in this interim analysis for effectiveness (retrospective, n = 150; prospective, n = 71).More than 80% of patients initiated reimbursed ibrutinib, with the remainder participating in the Medical Need Program.The median age at ibrutinib initiation was 72 (range 38-90) years, 64.3% of patients were male, 86.6% had an Eastern Cooperative Oncology Group performance status of 0-1, and 17.8% had a history of significant cardiovascular (CV) disease. Ongoing atrial fibrillation (AF) and other ongoing CV disease were both reported by 14/38 patients.Table 1 details the patient characteristics at baseline.The median time from diagnosis to ibrutinib initiation was 5.8 years and 72.6% patients had at least one prior line of therapy.In total, 78 of 132 assessed patients (59.1%) had del17p and/or TP53 mutation, and 72.5% of only 80 patients assessed had unmutated IGHV.In line with reimbursement criteria, 43.6% of patients with del17p and/or TP53 mutations had no prior lines of therapy, whereas 71.7% of patients without del17p and TP53 mutations had at least one prior line of therapy. The majority of patients (88.1%) initiated ibrutinib therapy at the recommended dose of 420 mg daily. ", "section_name": "Patient Demographics and Baseline Characteristics", "section_num": "3.1" }, { "section_content": "The median (range) follow-up was 34.3 (0.2-69.9) months in the total population.Table 2 details the main effectiveness outcomes.The cumulative ORR (best response at 60 months) was 90.0% (CR 16.7%; PR 51.6%; PR with lymphocytosis, 21.7%).The median (Q1-Q3) PFS for CLL was 38.3 (12.2-66.4)months for the total population, not estimable (NE) (15.7-NE) in the prospective group and 51.5 (29.1-NE) months in the retrospective group (Fig. 1).PFS was longer in patients without del17p and TP53 mutations than in those with del17p and/or TP53 mutations (49.9 versus 41.5 months; Table 2).There was little difference in PFS in patients with mutated or unmutated IGHV (49.9 versus 53.5 months).PFS varied slightly by line of therapy, but the variation did not follow any particular pattern.PFS was longer in patients younger than 65 years compared with those older than 65 years (49.5 versus 36.8 months).Median (Q1-Q3) OS was not estimable (21.3-NE) in the total population and was also not estimable in the prospective and retrospective groups (Fig. 2).The 36-month OS rate was 62.9% overall and 60.6% and 76.3% in the prospective and retrospective groups, respectively.Median time to next treatment was 35.7 months overall and not estimable and 51.5 months in the prospective and retrospective groups, respectively.The numbers obtained for the total population are a result of the statistical analysis used, as detailed in the methods. ", "section_name": "Effectiveness", "section_num": "3.2" }, { "section_content": "There were 226 patients in the safety analysis population, 73 in the prospective group and 153 in the retrospective group.The median (range) duration of ibrutinib treatment in the total safety population was 25.5 (0. 12.8%), toxicity (n = 26; 11.5%), death (n = 21; 9.3%), other (n = 18; 8.0%), comorbidities (n = 6; 2.7%), physician preference (n = 5; 2.2%) and one each (0.4%) of concomitant medication, patient preference and surgery.None of the deaths were reported as sudden cardiac deaths, and the one cardiac-related death (cardiac arrest) reported was considered not related to treatment by the investigator.Only two deaths were considered very likely related to ibrutinib by the investigators, pneumonia and aspergillus infection. As TEAEs consisting of all events related and unrelated to treatment that occurred during the study were reported for the prospective group, and only adverse events considered related to ibrutinib were reported for the retrospective group, they are reported separately for the two patient groups.TEAEs of interest are detailed in Table 3.For the prospective and retrospective groups, respectively, 100% and 83.7% of patients had at least one TEAE, while 58.9% and 43.8% of patients had at least one serious TEAE.In the prospective and retrospective groups, respectively, TEAEs led to ibrutinib dose reduction in 21.9% and 10.5% of patients, dose interruption in 39.7% and 23.5%, and withdrawal (which includes any discontinuations related or unrelated to ibrutinib therapy, including death, toxicity, comorbidities, progressive disease and physician preference) in 26.0% and 19.6%.Some patients were taking concomitant antithrombotic therapy during the study, 28 in the prospective and 42 in the retrospective group.Bleeding was more frequent in patients on such therapy compared with those not, 78.6% versus 31.1% in the prospective and 42.9% versus 25.2% in the retrospective group, and major bleeding was infrequent, 5.5% in the prospective group and 3.3% in the retrospective group.The most common antithrombotic therapy was an antiplatelet agent, 46.4% in the prospective group and 45.2% in the retrospective group, followed by a non-vitamin K oral anticoagulant, 10.7% in the prospective group and 21.4% in the retrospective group, vitamin K antagonist, none in the prospective group and 4.8% in the retrospective group, or other therapy, 14.2% in the prospective group and 9.5% in the retrospective group.More than one antithrombotic treatment was used by 28.6% of patients taking antithrombotic therapy in the prospective group and by 19.0% in the retrospective group; this included patients who switched therapies and, therefore, these patients were not all taking several antithrombotic agents concomitantly. ", "section_name": "Safety", "section_num": "3.3" }, { "section_content": "BiRD is an ongoing real-world study that aims to evaluate the effectiveness and safety of ibrutinib in patients with CLL in Belgium.Those enrolled in BiRD represent the clinical spectrum of a real-world CLL population, as they were mostly elderly (median age 72 years), and approximately three quarters had been treated previously.Almost half of patients with del17p and/or TP53 mutations had no prior lines of therapy, yet nearly three quarters of patients without del17p and TP53 mutations had at least one prior line of therapy, which is driven by the indication and reimbursement criteria in Belgium.It is encouraging that the vast majority of patients (88%) in this real-world population in normal clinical practice received the recommended dose of ibrutinib at treatment initiation.As many patients were included during the early years of ibrutinib market authorization, it is possible that physicians were initially cautious with dosing, starting at a dose lower than recommended, and titrating up to ensure tolerability. In this third interim analysis, the median follow-up for effectiveness was 34.3 months although there was a wide range, up to 70 months.The cumulative ORR up to 60 months was 90.0%, a finding similar to the ORR reported in the second interim analysis at 12 months (86.9%) [9], and also similar to some clinical trials with high-risk populations.In the RESONATE™ trial of single-agent ibrutinib in patients with R/R CLL, which had a comparable percentage of high-risk patients (32% with del17p, 51% with TP53 mutation; 86% considered high risk in the ibrutinib arm), the cumulative ORR for ibrutinib at the 6-year follow-up was 91% [14], and in a phase 2 study with high-risk patients (63% with del17p and/or TP53 mutation), the ORR for ibrutinib was 96% at a follow-up of 6 months [15].High rates of response have also been noted with ibrutinib treatment in clinical trials without such a high-risk population, such as the RESONATE-2™ study in previously untreated patients without del17p over the age of 65, which reported an ORR of 92% with up to 66 months follow-up [16].Other realworld studies have also produced similar levels of response to ibrutinib, including the FIRE real-world study, which is a similar design to our BiRD study, yet conducted in France.FIRE reported an ORR of 89.6% in the overall CLL patient group at a follow-up of 21.6 months in a population of patients with 58.7% del17p and/or TP53 mutations [17].In addition, a Danish retrospective cohort study in 205 patients, 72.1% of whom had del17p or TP53 mutations, with a median follow-up of 21.4 months, reported an ORR of 76.4% [18], and an analysis of 95 Swedish patients, 62% with del17p or TP53 mutations, treated with ibrutinib in a compassionate use program reported an ORR of 84% [19]. In our study, the median PFS was 38.3 months for the total population, which is unchanged from the second interim analysis at a median follow-up of 20.9 months [9].Interestingly, the PFS values for the separate prospective Table 3 Treatment-emergent adverse events (TEAE) of interest TEAE treatment-emergent adverse event a For retrospectively included patients, only reported ibrutinib-related adverse events were recorded from the period prior to inclusion in the study b Severe TEAE: on a scale of mild/moderate/severe and usually requires medical assistance/intervention/therapy and may require hospitalization c Serious TEAE: life threatening or causing death d Withdrawal includes any discontinuations that are related or unrelated to ibrutinib therapy, including death, toxicity, comorbidities, progressive disease and physician preference e Major bleeding is a severe/serious bleeding event f Percentages are calculated based on the numbers of patients with and without concomitant antithrombotic therapy in the retrospective group (42 and 111, respectively) and in the prospective group (28 and 45, respectively) and retrospective groups were both longer than this value (NE and 51.5 months, respectively), which is a result of the statistical analysis on the total cohort.As those patients in the retrospective group who died prior to study entry could not be included because they could not provide consent, an adjustment was made with left truncation to avoid bias in the retrospectively included patients on the total population analysis.As the retrospective group is larger than the prospective group of patients, this impacts the results for the total CLL cohort.The PFS values reported in our analysis are all within the range of those reported in clinical trials, such as RESONATE™, with a PFS of 44.1 months at 6 years' follow-up [14], and RESONATE-2™, with a PFS not estimable after a median follow-up of 60 months [16].In the FIRE real-world study of similar design, the overall PFS at median follow-up of 21.6 months was 37.7 months, and was not estimable in either the prospective or retrospective patient groups at 15.2 and 29.6 months' median follow-up, respectively [17].In the Danish real-world study, the median PFS was 41.2 months [18].PFS was shorter in patients with del17p and TP53 mutations (42 months compared with 50 months), yet this compares favorably with the PFS of 11 months reported for FCR treatment in previously untreated patients with TP53 alterations after 5 years' follow-up [20].Poor responses to chemotherapy in these high-risk patients have been reported previously [21], yet ibrutinib therapy is associated with durable responses [15,22].In patients with mutated or unmutated IGHV, the PFS was similar, as reported previously for ibrutinib [16]; however, only 60% of patients in our study underwent cytogenetic testing for del17p or TP53 and even fewer (36%) were tested for IGHV (it must be noted, though, that IGHV testing was not reimbursed for patients over 65 years of age in Belgium at the start of the study, and is currently only reimbursed for patients eligible for chemoimmunotherapy treatment).This highlights a potentially important gap in testing, a finding also noted in another real-world study in the United States [23], which could increase the likelihood of patients not receiving optimal treatment in clinical practice.Cytogenetic testing is now becoming more common, and this will help to improve treatment choices. As expected, younger patients (aged < 65 years) had longer PFS than those aged ≥ 65 years, and median PFS varied slightly by number of previous lines of therapy.This variation did not follow any particular pattern, partly due to low patient numbers in each group and the long PFS values at this time of follow-up.Interestingly, this non-correlation of PFS with prior therapy line was also noted in a retrospective US real-world cohort analysis [24].Patients were enrolled into our study at the beginning of ibrutinib use, when practice policies were adapted, and it is therefore likely that early recruits were of higher risk than later ones, and that better results may be expected with longer follow-up, particularly in previously untreated patients. The median OS in our study was not estimable, as also reported in several other studies at time points shorter than several years [7,8,16,17].The estimated 36-month OS rate of 62.9% is in the expected range, when considering the OS rate of 63% at 30 months in the Swedish cohort study [19] and in other real-world studies reporting at earlier time points: the Danish cohort study showed an estimated OS of 76.8% at 24 months [18], and the FIRE study estimated a value of 86.9% at the same 24-month time point [17].It is likely that further follow-up is needed to determine additional information on survival. In the safety analysis with up to 70 months of follow-up, fewer TEAEs were reported in the retrospective than in the prospective group, as only events considered related to ibrutinib were reported retrospectively.In general, TEAEs were in the range of those previously reported in real-world studies, including FIRE [17], the Denmark cohort [18] and the Swedish cohort [19], and within the range of single-agent ibrutinib clinical trials of CLL [2,3,14].Patients treated in a real-world setting, however, may be expected to have higher rates of TEAEs and treatment discontinuation than those in a clinical trial setting, because a real-world population includes higher risk patients who might be excluded from clinical trials.The rate of discontinuations due to toxicity observed in our study (11.5%) was lower than that reported in other real-world studies (20% in the Swedish retrospective study [19], 22.9% in the Danish retrospective study [18], 20.8% in a US cohort study [24] and 17.5% in a large UK real-world study from 2016 of 315 patients after 16 months median follow-up [25]).These older studies, however, may have included higher-risk populations in the earlier days of ibrutinib use, particularly with compassionate use, potentially leading to higher discontinuations.The frequency of bleeding in our study was similar to that experienced in clinical trials and real-world studies [7,8,16,17].Bleeding was more frequent in patients receiving antithrombotic therapy, but major bleeding events were too infrequent to determine any correlation.The frequency of TEAEs varied little from the second [10] to the third interim analysis of BiRD, which is to be expected as TEAEs are reported by actual study time and not by treatment course per patient.It is reassuring that no cardiac deaths or sudden deaths were reported during this long follow-up period.Overall, treatment with ibrutinib was tolerable and no new safety signals were observed. There are a number of limitations to consider when evaluating the results from this analysis.Unlike clinical trials, the effectiveness and safety parameters are presented through descriptive data in a real-world setting and assessed by the investigators.The response was assessed by physicians in routine clinical practice, and therefore comparison with clinical trials is challenging.The differences between the retrospective and prospective populations are evident, and adjustments to the overall population aimed to reduce bias, although bias cannot be entirely discounted. This large, real-world study gives a unique perspective of ibrutinib use in a single country, encompassing most major hematology centers in Belgium.It provides information on the long-term use of ibrutinib in CLL in real-world clinical practice, with up to 7 years (median 34 months) follow-up.It is promising to see that results from randomized trials in more restricted populations have translated into real world with ibrutinib. ", "section_name": "Discussion", "section_num": "4" }, { "section_content": "Results from this large, real-world study show ibrutinib to be effective in this mostly elderly CLL population, with results in alignment with those from randomized controlled trials and with other real-world studies.TEAEs were in line with those from other studies, few patients discontinued due to toxicity and no new safety signals were evident.We await further follow-up from the BiRD study to confirm the longer-term effectiveness and safety of ibrutinib. ", "section_name": "Conclusions", "section_num": "5" } ]	[ { "section_content": "The authors would like to thank all the patients who were included in this analysis.Writing assistance was provided by Emma Fulkes, Ph.D., of Parexel, and was funded by Janssen Research & Development.Statistical support was provided by Simon Paternotte of ICTA and funded by Janssen Research and Development. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Funding This study was sponsored by Janssen Pharmaceutica NV, and Pharmacyclics LLC, an AbbVie Company.Writing assistance was provided by Emma Fulkes of Parexel and funded by Janssen Global Services, LLC. ", "section_name": "", "section_num": "" }, { "section_content": "The data-sharing policy of Janssen Pharmaceutical Companies of Johnson & Johnson is available at https:// www.janss en.com/ clini cal-trials/ trans paren cy.As noted on this site, requests for access to the study data can be submitted through Yale Open Data Access (YODA) Project site at http:// yoda.yale.edu. ", "section_name": "Availability of Data and Material", "section_num": null }, { "section_content": "Author Contributions All authors reviewed and approved the manuscript.AJ, MA, BDB and CVB also provided study design and RW also provided data analysis. Conflict of Interest AJ reports consultancy for Janssen, Roche, Gilead, AbbVie, Novartis, Amgen, Sanofi-Genzyme and Celgene; research grant from Janssen; travel grant from Janssen, Celgene, AbbVie and Roche.ZNB reports research grants from Janssen, Roche, Takeda, CAF-DCF.SM reports consultancy for Janssen.DB reports consultancy and research grant from Janssen.FO The study protocol was approved by the institutional review boards or independent ethics committees of participating centers.All patients received oral and written information on the study and provided informed consent to data collection and source data verification. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http:// creat iveco mmons.org/ licen ses/ by/4.0/. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Declarations", "section_num": null }, { "section_content": "Author Contributions All authors reviewed and approved the manuscript.AJ, MA, BDB and CVB also provided study design and RW also provided data analysis. ", "section_name": "", "section_num": "" }, { "section_content": "Conflict of Interest AJ reports consultancy for Janssen, Roche, Gilead, AbbVie, Novartis, Amgen, Sanofi-Genzyme and Celgene; research grant from Janssen; travel grant from Janssen, Celgene, AbbVie and Roche.ZNB reports research grants from Janssen, Roche, Takeda, CAF-DCF.SM reports consultancy for Janssen.DB reports consultancy and research grant from Janssen.FO ", "section_name": "Declarations", "section_num": null }, { "section_content": "The study protocol was approved by the institutional review boards or independent ethics committees of participating centers.All patients received oral and written information on the study and provided informed consent to data collection and source data verification. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http:// creat iveco mmons.org/ licen ses/ by/4.0/. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Ethical Approval", "section_num": null } ]
10.3390/reports7030071	Assessing the Methylation Status of Two Potential Key Factors Involved in Cervical Oncogenesis	<jats:p>(1) Background: Cervical cancer, caused mainly by high-risk Human Papillomavirus (hrHPV), is a significant global health issue. While a Pap smear remains a reliable method for early detection, identifying new biomarkers to stratify the risk is crucial. For this purpose, extensive research has been conducted on detecting DNA methylation. (2) Methods: This cross-sectional study aimed to assess the expression levels of EIF4G3 and SF3B1 in precursor lesions and cervical tumor tissues through qRT-PCR and evaluate the methylation status of their promoters through bisulfite conversion. (3) Results: Both genes showed similar mRNA expression patterns, with the highest levels observed in squamous cell carcinoma (SCC) samples (p < 0.0001). Additionally, methylation analysis indicated increased percentages in the control group for both factors. Notably, the expression levels of both genes were inversely correlated with promoter methylation (EIF4G3—p = 0.0016; SF3B1—p < 0.0001). (4) Conclusions: Regarding the methylation pattern for both genes, we observe a decreasing trend from NILM to SCC patients. Therefore, we concluded that the decrease in methylation at the promoter level for both genes could be an indicator of abnormal cytology.</jats:p>	[ { "section_content": "Cervical cancer remains one of the most prevalent cancers among women globally and continues to pose a significant clinical and societal burden, particularly in resource-limited countries.According to the last statistics of GLOBOCAN from 2022, this is the fourth most common cancer among women (incidence of 6.8%), following breast, colorectal, and lung cancer.In addition, it is the most common cancer type in 25 countries and the leading cause of cancer death in 37 countries [1].Cervical carcinogenesis progresses stepwise, beginning with HPV infection and advancing from premalignant stages to invasive cancer over at least a decade.Following a persistent infection with a hr-HPV, further viral-induced genetic and epigenetic alterations in the host cell genome are crucial for the development of cervical cancer [2]. Presently, the prevention and diagnosis of cervical cancer rely on cytological (Papanicolaou test) and histopathological examinations.The Pap smear is among the most reliable methods for early detection of cervical cancer (CC) and is considered the gold standard diagnostic test for asymptomatic women.In well-established healthcare systems, it can reduce the average annual mortality rate by 2.6% [3].Some studies revealed a link between cervical cancer and some of its high-grade precursor lesions and elevated DNA methylation levels of numerous tumor suppressor genes [4].Therefore, identifying new potential biomarkers to discern women at risk of developing cervical cancer is crucial.Furthermore, Reports 2024, 7, 71 2 of 12 abnormal DNA methylation can occur in low-grade intraepithelial lesions (LGSIL), suggesting its potential application in the early diagnosis of cervical cancer.Detecting altered DNA methylation at this stage is very important, as LGSIL can either regress or progress to higher-grade lesions [5]. Currently, DNA methylation of the most investigated sites potentially associated with identifying high-grade cervical disease with good sensitivity and specificity includes CADM1, MAL, miR-124a, EPB41L3, JAM3, TERT, C13ORF18, LMX1, SOX1, PAX1, and NKX6-1 [6][7][8].Moreover, some of these genes are included in commercial DNA methylation tests, which offer the advantage of using tissue samples and other body fluids (e.g., liquid biopsy).In the context of cervical cancer, these assays can be used alone or combined with traditional screening methods to enhance triage and therapy management, providing valuable insights into epigenetic profiles [9]. Using ChIP-sequencing approaches, we previously identified nine genes as potential biomarkers, including EIF4G3 (Eukaryotic translation initiation factor 4 γ 3) and SF3B1 (Splicing factor 3b subunit 1) that are involved in similar putative pathways interacting with the same factors [10].In this context, this study aimed to evaluate the potential of the investigated factors EIF4G3 and SF3B1, including the methylation status of CpG islands around gene promoters and their expression in cervical cancer and precursor lesions, and to assess their prognostic potential. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "", "section_name": "Materials and Methods", "section_num": "2." }, { "section_content": "Cervical samples consisted of 52 HPV-positive cervical cytology specimens and tumor tissue from squamous cervical carcinomas (SCC) selected from a total of 110 women who self-referred for gynecological examinations \"Cuza Voda\" Clinical Hospital of Obstetrics and Gynecology, Iasi.The inclusion criteria for this study were women aged 18 and above who were not currently pregnant.Participants needed to have abstained from vaginal contact or showers for at least three days prior to sampling.The control group consisted of cervical specimens from women with negative cytology without HPV infection.This study followed the principles of the Declaration of Helsinki, and all participants provided written informed consent before taking part. For the Papanicolaou and HPV genotyping test, a cervical sample was collected using a separate Cervex Brush (Avantor, Radnor, PA, USA).The liquid-based preparation method for collecting cervicovaginal samples was performed according to the manufacturer's instructions (ThinPrep-Hologic, Bedford, MA, USA), and the samples were stored until further analysis.The samples were collected and preserved for DNA methylation analysis using an ESwab (COPAN, Brescia, Italy).(COPAN, Brescia, Italy).These specimens were stored at -80 • C until they were utilized.All samples for these assays were collected during a single visit. Cytology diagnoses were made according to the Bethesda System grading criteria.Based on these management guidelines, selected patients were recommended to undergo colposcopy and either a Punch Biopsy (PB) or a Large Loop Excision of the Transformation Zone (LLETZ).Histology results were classified as no dysplasia, CIN grade I, II, III, or cervical cancer. ", "section_name": "Patients and Samples Collection", "section_num": "2.1." }, { "section_content": "DNA was isolated from cervical specimens using the QIAamp DNA Mini Kit (Qiagen) following the producer's guidelines.The concentration and purity of each DNA sample were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). ", "section_name": "DNA Isolation", "section_num": "2.2." }, { "section_content": "Human Papillomavirus (HPV) detection and genotyping were conducted for all samples using the INNO-LiPA ® HPV Genotyping Extra II kit (Fujirebio Europe, Ghent, Belgium) Reports 2024, 7, 71 3 of 12 following the manufacturer's instructions.The method allows the classification of samples into high-risk (hrHPV), low-risk (lrHPV), and undetermined-risk HPV types.The test specifically detects 13 high-risk HPV types: 16,18,31,33,35,39,45,51,52,56,58,59, and 68. ", "section_name": "HPV DNA Detection and Genotyping", "section_num": "2.3." }, { "section_content": "Bisulfite conversion was carried out using the EpiTect Bisulfite kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol.An input of 700 ng of the DNA sample in a total volume of 20 µL was converted, along with positive and negative controls (CpGenome Universal Methylated/Unmethylated DNA) (Millipore, Billerica, MA, USA). ", "section_name": "Bisulfite Conversion", "section_num": "2.4." }, { "section_content": "The extraction of total RNA was performed for all samples using TriZol reagent (Invitrogen, Carlsbad, CA, USA) and the purification with RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.Total RNA samples were subsequently reverse-transcribed into cDNA utilizing the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc.) using an input of 1 µg of each RNA sample. ", "section_name": "RNA Isolation and cDNA Synthesis", "section_num": "2.5." }, { "section_content": "Specific primers for the targeted genes were designed using the Primer-BLAST tool (www.ncbi.nlm.nih.gov/tools/primer-blast/,accessed on 23 February 2024).These qPCR data were analyzed, and relative expression was calculated using the quantification cycle (Cq) with the 2 -∆Cq /2 -∆∆Cq method.Methylation primers were designed using the Meth-Primer algorithm (http://www.urogene.org/methprimer/,accessed on 12 March 2024), which predicts CpG islands defined as 200 bp DNA sequences with a GC content greater than 50% [11].The primers used in this study were synthesized by Biolegio (Nijmegen, The Netherlands).They were designed to distinguish between methylated and unmethylated DNA following bisulfite treatment.The sequences of all the primers used, along with their respective parameters, can be found in Table 1.For mRNA expression levels detection, qRT-PCR has been performed on Applied Biosystems 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA), using the GAPDH gene as a reference gene.The experiments were measured in triplicate, and relative expression was determined using the 2 -∆Cq /2 -∆∆Cq method [12].To assess the degree of methylation in the samples, direct quantitative methylation-specific PCR (qMSP) was performed on genomic DNA.Standard curves were created with serially diluted positive (fully methylated) and negative (fully unmethylated) controls at concentrations of 50 pg, 500 pg, 5 ng, and 50 ng. The qRT-PCR and qMS-PCR were conducted in a final volume of 25 µL, which included 12.5 µL of Maxima SYBR Green/ROX qPCR Master Mix (2X) (Thermo Fisher Scientific, Waltham, MA, USA), 0.30 µM for each primer, and 50 ng of target (cDNA, respectively bisulfite-treated DNA).The methylation percentage (%M) was calculated using the formula described by Fackler et al. (% methylation = 100 • [ng methylated gene A/(ng methylated gene A + ng unmethylated gene A)] [13].The concentration of unmethylated (U) and methylated (M) DNA for each patient sample was extrapolated using the standard curves. ", "section_name": "Primer Design", "section_num": "2.6." }, { "section_content": "The statistical analysis was performed with GraphPad Prism version 9.3 software (Graph Pad Software Inc., San Diego, CA, USA).To evaluate if the data sets were normally distributed, we applied the Shapiro-Wilk test, which is an appropriate method for small sample sizes (<50 samples).When p > 0.05, the null hypothesis is accepted, and data are called normally distributed.A simple linear regression test was used to evaluate the correlation between expression levels and gene methylation status.Moreover, the t-test parametric and Mann-Whitney non-parametric tests were used to compare study groups when appropriate.p-values < 0.05 were considered statistically significant. ", "section_name": "Statistical Analysis", "section_num": "2.8." }, { "section_content": "", "section_name": "Results", "section_num": "3." }, { "section_content": "Samples from patients (n = 62) were divided into six groups according to their Papanicolaou test results and the presence or absence of HPV.The HPV-positive samples were classified in: 16.13% LGSIL (Low-Grade Squamous Intraepithelial Lesion) (n = 10), 16.3% HGSIL (High-Grade Squamous Intraepithelial Lesion) (n = 10), ASCUS (Atypical Squamous Cells of Undetermined Significance) (n = 10), 12.91% ASCH (Atypical Squamous Cells) (n = 8) and 22,58% tissue specimens from squamous cervical carcinomas (SCCs) (n = 14).The control group consisted of samples (16.13%) identified as Negative for Intraepithelial Lesion or Malignancy and negative for HPV (NILM-) (n = 10).In selected cases, targeted biopsies were performed following colposcopy for further evaluation.Notably, the biopsy results did not alter the LSIL, HGSIL cytological groups, and SCC classification.Regarding the ASCUS and ASCH groups, we selected the patients who underwent biopsies, and the histology results were more heterogeneous.The ASCUS patients included in this study presented 60% (6/10) CINI, 30% (3/10) CINII, and 10% CINIII.In the ASCH group, the most prevalent was CINIII diagnosis-50% (4/8), followed by CINII-37.5% (3/8), and CINI-12.5% (1/8). In terms of HPV genotype diversity, we observed that the LGSIL and AS-CUS groups showed the greatest variety, with over 10 genotypes present among the samples from these groups.In contrast, the SCC and HGSIL groups showed the least diversity, identifying only six genotypes.Notably, all patients in the SCC group had single HPV infections, specifically HPV16, HPV18, HPV45, and HPV52 (Figure 1).Furthermore, we observed that HPV16 had the highest prevalence, accounting for 23.81% of the total investigated samples.Prevalence data are shown in Figure 2. ", "section_name": "Study Group Characterization", "section_num": "3.1." }, { "section_content": "Upon evaluating the distribution of our data sets from the studied groups using the Shapiro-Wilk normality test, we found that all values for EIF4G3 were normally distributed except in the SCC group.For SF3B1, the SCC and HGSIL groups deviated from normality.In these instances, the Mann-Whitney test was applied (Supplementary Table S1).When investigating the methylation status of both studied genes, we found that the highest percentage of methylation was in control samples, with medians of 88.76% (range: 81.20-98.35%)for EIF4G3 and 87.53% (range: 78.95-98.22%)for SF3B1.In the EIF4G3 promoter, CpG islands showed increased methylation percentages in ASCUS and LGSIL patients, with medians of 42.83% and 36.23%,respectively, but still lower than the control group.Samples from ASCH and HGSIL exhibited similar patterns, with median values of Furthermore, we observed that HPV16 had the highest prevalence, accounting for 23.81% of the total investigated samples.Prevalence data are shown in Figure 2. ", "section_name": "Evaluation of Promoter Methylation Status", "section_num": "3.2." }, { "section_content": "Upon evaluating the distribution of our data sets from the studied groups using the Shapiro-Wilk normality test, we found that all values for EIF4G3 were normally distributed except in the SCC group.For SF3B1, the SCC and HGSIL groups deviated from normality.In these instances, the Mann-Whitney test was applied (Supplementary Table S1).When investigating the methylation status of both studied genes, we found that the highest percentage of methylation was in control samples, with medians of 88.76% (range: 81.20-98.35%)for EIF4G3 and 87.53% (range: 78.95-98.22%)for SF3B1.In the EIF4G3 promoter, CpG islands showed increased methylation percentages in ASCUS and LGSIL patients, with medians of 42.83% and 36.23%,respectively, but still lower than the control group.Samples from ASCH and HGSIL exhibited similar patterns, with median values of ", "section_name": "Evaluation of Promoter Methylation Status", "section_num": "3.2." }, { "section_content": "Upon evaluating the distribution of our data sets from the studied groups using the Shapiro-Wilk normality test, we found that all values for EIF4G3 were normally distributed except in the SCC group.For SF3B1, the SCC and HGSIL groups deviated from normality.In these instances, the Mann-Whitney test was applied (Supplementary Table S1).When investigating the methylation status of both studied genes, we found that the highest percentage of methylation was in control samples, with medians of 88.76% (range: 81.20-98.35%)for EIF4G3 and 87.53% (range: 78.95-98.22%)for SF3B1.In the EIF4G3 promoter, CpG islands showed increased methylation percentages in ASCUS and LGSIL patients, with medians of 42.83% and 36.23%,respectively, but still lower than the control group.Samples from ASCH and HGSIL exhibited similar patterns, with median values of 13.22% and 15.21%, respectively, and methylation percentages ranging from 3.31 to 25.57% and 6.39 to 22.56%.The lowest values were observed in SCC tissue samples, with a median of 1.49% Table 2.For the SF3B1 promoter, the lowest methylation values were found in SCC samples (0-3.08%),followed by slight increases in the HGSIL (range: 38.61-73.89%)and ASCH (6.43-16.50%)groups.Higher percentages were observed in the LGSIL (15.97-36.60%)and ASCUS (38.61-73.89%)lesions. All results indicate a significantly decreased percentage of promoter methylation in all studied groups compared with controls (p < 0.0001).The methylation profiles of EIF4G3 and SF3B1 in the studied groups are presented in Figure 3.For the SF3B1 promoter, the lowest methylation values were found in SCC sam (0-3.08%),followed by slight increases in the HGSIL (range: 38.61-73.89%)and A (6.43-16.50%)groups.Higher percentages were observed in the LGSIL (15.97-36.60%)ASCUS (38.61-73.89%)lesions. All results indicate a significantly decreased percentage of promoter methylatio all studied groups compared with controls (p < 0.0001).The methylation profiles of EIF and SF3B1 in the studied groups are presented in Figure 3.For both genes, a comparison of methylation levels between ASCUS and HG showed a significant difference, with lower levels observed in HGSIL patients (p < 0.0 Additionally, comparing the ASCH group with the SCC group revealed significant di ences for both EIF4G3 (p = 0.0004) and SF3B1 (p < 0.0001).However, no statistical d ence was found between the ASCH and HGSIL groups.The biopsy results showed 60% of patients with ASCH presented a CINIII diagnosis, with this group presenti similar percent of methylation level with HGSIL.For both genes, a comparison of methylation levels between ASCUS and HGSIL showed a significant difference, with lower levels observed in HGSIL patients (p < 0.0001).Additionally, comparing the ASCH group with the SCC group revealed significant differences for both EIF4G3 (p = 0.0004) and SF3B1 (p < 0.0001).However, no statistical difference was found between the ASCH and HGSIL groups.The biopsy results showed that 60% of patients with ASCH presented a CINIII diagnosis, with this group presenting a similar percent of methylation level with HGSIL. ", "section_name": "Evaluation of Promoter Methylation Status", "section_num": "3.2." }, { "section_content": "After evaluating the distribution of gene expression values across the studied groups using the Shapiro-Wilk normality test, we found that all values adhered to a normal distribution (Supplementary Table S1).The qRT-PCR results indicated that mRNA expression levels of both EIF4G3 and SF3B1 genes were significantly higher in all studied groups compared with the control group, except for the ASCH group.Moreover, the SCC group exhibited the most significant increases for both genes (p < 0.0001), with mean values of -2.227 and -1.540, respectively, compared with the NILM (-) group, which had mean values of -4.649 and -4.242.Additionally, significant results were observed for the EIF4G3 gene in the ASCUS group (p < 0.0001, median = -2.221)and for the SF3B1 gene in the LGSIL group (p < 0.0001, median = -2.220)when compared with the control group Table 3.The gene expression profiles of EIF4G3 and SF3B1 in the studied groups are presented in Figure 4. Reports 2024, 7, x FOR PEER REVIEW 7 of 12 ", "section_name": "Evaluation of Gene Expression Levels in Patient Samples and Correlation between Expression Levels and Methylation Percentage", "section_num": "3.3." }, { "section_content": "After evaluating the distribution of gene expression values across the studied groups using the Shapiro-Wilk normality test, we found that all values adhered to a normal distribution (Supplementary Table S1).The qRT-PCR results indicated that mRNA expression levels of both EIF4G3 and SF3B1 genes were significantly higher in all studied groups compared with the control group, except for the ASCH group.Moreover, the SCC group exhibited the most significant increases for both genes (p < 0.0001), with mean values of -2.227 and -1.540, respectively, compared with the NILM (-) group, which had mean values of -4.649 and -4.242.Additionally, significant results were observed for the EIF4G3 gene in the ASCUS group (p < 0.0001, median = -2.221)and for the SF3B1 gene in the LGSIL group (p < 0.0001, median = -2.220)when compared with the control group Table 3.The gene expression profiles of EIF4G3 and SF3B1 in the studied groups are presented in Figure 4.When comparing the expression levels between the ASCH and SCC groups, there was a significantly higher expression of both genes in SCC patients (p = 0.0102 for EIF4G3 and p < 0.0001 for SF3B1).In the comparison between the ASCH and HGSIL groups, a significant increase in expression was observed for the SF3B1 gene in the HGSIL group.Conversely, when comparing the ASCUS and HGSIL groups, only EIF4G3 expression When comparing the expression levels between the ASCH and SCC groups, there was a significantly higher expression of both genes in SCC patients (p = 0.0102 for EIF4G3 and p < 0.0001 for SF3B1).In the comparison between the ASCH and HGSIL groups, a significant increase in expression was observed for the SF3B1 gene in the HGSIL group.Conversely, when comparing the ASCUS and HGSIL groups, only EIF4G3 expression levels were significantly higher in HGSIL patients (p = 0.0033).It seems that the expression level of the SF3B1 gene could better discriminate between the ASCH and SCC groups. Further, we investigated the correlation between mRNA expression levels and methylation status for both genes and observed a significant inverse correlation.For EIF4G3, the correlation had a p-value of 0.0016 (Y = -0.01252X-2.543), and for SF3B1, the p-value was less than 0.0001 (Y = -0.01782X-2.240) (Figure 5).levels were significantly higher in HGSIL patients (p = 0.0033).It seems that the expression level of the SF3B1 gene could better discriminate between the ASCH and SCC groups. Further, we investigated the correlation between mRNA expression levels and methylation status for both genes and observed a significant inverse correlation.For EIF4G3, the correlation had a p-value of 0.0016 (Y = -0.01252X-2.543), and for SF3B1, the p-value was less than 0.0001 (Y = -0.01782X-2.240) (Figure 5). ", "section_name": "Evaluation of Gene Expression Levels in Patient Samples and Correlation between Expression Levels and Methylation Percentage", "section_num": "3.3." }, { "section_content": "Testing for high-risk HPV (hrHPV) DNA with new molecular instruments demonstrates excellent performance and reproducibility.Cuzick et al. show that HPV testing has a sensitivity of 90-100% for detecting precancerous lesions, compared with a sensitivity of 50-80% for cytological screening [14,15].Research in the field of epigenetics has demonstrated that aberrant DNA methylation is a common alteration in cancer [16].The hypermethylation of specific DNA regions during carcinogenesis could serve as a sensitive screening tool, particularly because different methylation patterns of tumor suppressor genes have been identified in HPV-induced tumors [17].Methylation markers are valuable in cervical cancer screening programs, with studies showing they have higher specificity compared with HPV testing and immunohistochemistry (p16/Ki-67) [18].These markers can be utilized not only in tissue samples but also in any body fluid (liquid biopsy).Here are several methylation kits currently available on the market.The QIAsure Methylation Test (Qiagen, Hilden, Germany) is a multiplex quantitative methylation-specific PCR (qMSP)-based assay that amplifies the methylated promoter regions of the FAM19A4/miR-124-2 genes, showing increased sensitivity for identifying advanced transforming CIN3+ (69.4-77.8%)and cervical cancer (100%) in hrHPV-positive samples [19].Another MSP-based assay, GynTect ® (Oncgnostics, Jena, Germany), distinguishes between cervical lesion types by examining the methylation status of the promoter regions ", "section_name": "Discussion", "section_num": "4." }, { "section_content": "Testing for high-risk HPV (hrHPV) DNA with new molecular instruments demonstrates excellent performance and reproducibility.Cuzick et al. show that HPV testing has a sensitivity of 90-100% for detecting precancerous lesions, compared with a sensitivity of 50-80% for cytological screening [14,15].Research in the field of epigenetics has demonstrated that aberrant DNA methylation is a common alteration in cancer [16].The hypermethylation of specific DNA regions during carcinogenesis could serve as a sensitive screening tool, particularly because different methylation patterns of tumor suppressor genes have been identified in HPV-induced tumors [17].Methylation markers are valuable in cervical cancer screening programs, with studies showing they have higher specificity compared with HPV testing and immunohistochemistry (p16/Ki-67) [18].These markers can be utilized not only in tissue samples but also in any body fluid (liquid biopsy).Here are several methylation kits currently available on the market.The QIAsure Methylation Test (Qiagen, Hilden, Germany) is a multiplex quantitative methylation-specific PCR (qMSP)-based assay that amplifies the methylated promoter regions of the FAM19A4/miR-124-2 genes, showing increased sensitivity for identifying advanced transforming CIN3+ (69.4-77.8%)and cervical cancer (100%) in hrHPV-positive samples [19].Another MSPbased assay, GynTect ® (Oncgnostics, Jena, Germany), distinguishes between cervical lesion types by examining the methylation status of the promoter regions of six genes (astrotactin1 (ASTN1), distal-less homeobox 1 (DLX1), integrin subunit α 4 (ITGA4), relaxin family peptide receptor 3 (RXFP3), SRY-Box Transcription Factor 17 (SOX17), and zinc finger protein 671 (ZNF671)) and uses two quality control markers (iduronate 2-sulfatase-M (ID2S) and acetylcholinesterase (AChE)).This assay showed a sensitivity for CIN3+ ranging from 31.6% to 67.7% and a specificity for <CIN3 ranging from 82.6% to 95.9% [20,21].Other methylation tests include Confidence Marker (Neumann Diagnostics, Budapest, Hungary), Cervi-M (Ingenuity Healthcare, Mumbai, India), Precursor-M Test ® (Self-screen B.V., Amsterdam, The Netherlands), PAX1 DNA Detection kit, ZNF582 DNA Detection kit (iStat Biomedical Co., Ltd., New Taipei City, Taiwan) and S5 classifier (S5 ® CareLYFE, Zhuhai, China) [21]. Therefore, we intended to evaluate further and validate the prospective prognostic potential of two significant genes, EIF4G3 and SF3B1, that we previously identified with increased mRNA expression levels in precursor lesions through lavage sample testing these markers in a cohort study (new patients) and determine the risk of progression of low-and high-grade CIN lesions [10]. The SF3B1 gene encodes the largest subunit of the splicing factor 3b protein complex, which is essential for spliceosome assembly and mRNA splicing.When the SF3B1 gene is mutated, it produces a protein that alters the normal mRNA processing mechanism, leading to the abnormal splicing and potential downregulation of numerous mRNAs [22].The involvement of this factor in cervical cancer development is unknown.However, mutations in the SF3B1 gene are the most common and significant among spliceosome mutations in hematological diseases [23].SF3B1 mutations are known to contribute to tumor pathogenesis by disrupting various cellular functions and pathways, including heme biosynthesis, mitochondrial metabolism, and the NF-κB pathway [24].SF3B1 is altered in approximately 15-20% of all myelodysplastic syndromes (MDS) patients, and this alteration increases to over 80% in MDS, specifically with ring sideroblasts (RS) [25].The presence of an SF3B1 mutation appears to be an early event in MDS pathogenesis, being linked to a unique gene expression profile, and is associated with a favorable prognosis and a low risk of progression to acute myeloid leukemia (AML) [26].In contrast to MDSs, SF3B1 mutations in myeloproliferative neoplasms (MPNs) seem to elevate the risk of fibrotic transformation [27].SF3B1 mutations are relatively uncommon in chronic myelomonocytic leukemia (CMML) patients, occurring in about 5-6% of cases, and similar to MDS, these mutations are associated with the RS phenotype [25,28,29].Simmler P et al. showed that the splicing factor SF3B1 is also frequently mutated in pancreatic ductal adenocarcinoma (PDAC), and SF3B1 K700E functions as an oncogenic driver in PDAC, promoting the advancement of early-stage tumors by hindering the cellular response to the tumor-suppressive effects of TGF-β [30].Popli P et al. revealed elevated SF3B1 protein expression in human endometrial tumors and three endometrial cancer cell lines, consistent with increased expression of other splicing factors observed in various human cancers [31].The in vitro experiments demonstrate that SF3B1 enhances endometrial cancer cell proliferation, cell cycle progression, migration, and invasion [32].This is the first study that indicated the potential involvement of SF3B1 in cervical cancer development as the expression levels were significantly elevated in cervical cancer samples compared with the control group, but also in precursor lesions.In our previous study, we showed that its expression is inhibited when E6 and E7 oncogenes are silenced because of global chromatin deposition of the MBD2/MBD3 NuRD complex [10].Notably, we observed significant hypomethylation of CpG islands in cervical cancer, which was significantly correlated with mRNA expression levels.This indicates that the gene promoter demethylation induced by hrHPV infection could be the main cause of increased gene expression levels.Given its high mutational rate, potential fusion with another gene, or overexpression, we could hypothesize its role as a proto-oncogene.In this context, we also examined the methylation status of gene promoters and found a progressive decrease from precancerous lesions to cervical cancer.Regarding the most heterogeneous group ASCUS and ASCH, we observed that the results from the methylation analysis discriminate better than the gene expression level between the different neoplasia types. A similar pattern to the SF3B1 factor was found for the eukaryotic translation initiation factor 4G (EIF4G3) gene.EIF4G3 is an important scaffold protein in the translation initiation complex.It is part of the EIF4F complex, essential for initiating protein synthesis by binding to the mRNA cap structure and recruiting the ribosome to the mRNA.In a mice study by Hu J et al., the mutation in the EIF4G3 gene was found to lead to male infertility due to meiotic arrest at the end of the meiotic prophase [33].Evaluating its mRNA expression levels, we concluded that they increase with the progression of precursor lesions to cervical cancer.Moreover, the investigation of gene promoters' methylation status revealed higher percentages in control groups versus precursor lesions or cervical cancer, and the expression levels also correlated with methylation status.Studies about the role of this factor in oncogenesis are scarce; therefore, this study is the first to report the involvement in cervical oncogenesis.However, this study has a limitation in terms of the number of patients included, but it could serve as a strong foundation for a larger investigation. ", "section_name": "Discussion", "section_num": "4." }, { "section_content": "Measuring methylation levels alongside gene expression levels could be a valuable tool for stratifying hr-HPV-positive patients with abnormal Pap tests, especially those with LGSIL, ASCUS, and ASCH cytology.It is well known that LGSIL may either regress or progress to more advanced stages of cervical cancer, while ASCUS and ASCH citologies are highly heterogeneous. This study highlights the importance of epigenetic changes, particularly aberrant methylation, in the development of cervical cancer.By identifying new factors such as SF3B1 and EIF4G3, which show increased mRNA expression and altered methylation patterns in cervical cancer and precursor lesions, the research underscores the potential of these genes as biomarkers for early detection and diagnostic precision.While the role of SF3B1 in cervical cancer development is newly reported here, its involvement in other cancers and cellular processes indicates its broader oncogenic potential.Similarly, EIF4G3's role in oncogenesis is underexplored, but this study establishes its relevance in cervical cancer progression. ", "section_name": "Conclusions", "section_num": "5." }, { "section_content": "The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/reports7030071/s1,Table S1: p-values from the Shapiro-Wilk test when assessing normality in the studied groups.p-value > 0.05. ", "section_name": "Supplementary Materials:", "section_num": null } ]	[ { "section_content": "We would like to thank our colleague Bunea Florentina for her valuable assistance in sample preparation.Additionally, we extend our appreciation to the reviewers for their time and effort in reviewing this manuscript. ", "section_name": "Acknowledgments:", "section_num": null }, { "section_content": "The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. ", "section_name": "Data Availability Statement:", "section_num": null }, { "section_content": "Funding: This research was funded by the Romanian Academy, Bucharest, Romania, and UEFISCDI, grant number PN-III-P1-1.1-TE-2019-1759(TE 39/2020). Institutional Review Board Statement: All procedures were conducted in accordance with the ethical standards of the responsible committee on human experimentation (both institutional and national) as outlined in the Helsinki Declaration of 1975, revised in 2000.This study was approved by the University of Medicine and Pharmacy \"Grigore T. Popa\" Ethics Committee, approval number 57/17.03.2021. Informed Consent Statement: Informed consent was obtained from all subjects involved in this study. The authors declare no conflicts of interest. ", "section_name": "Conflicts of Interest:", "section_num": null }, { "section_content": "Funding: This research was funded by the Romanian Academy, Bucharest, Romania, and UEFISCDI, grant number PN-III-P1-1.1-TE-2019-1759(TE 39/2020). Institutional Review Board Statement: All procedures were conducted in accordance with the ethical standards of the responsible committee on human experimentation (both institutional and national) as outlined in the Helsinki Declaration of 1975, revised in 2000.This study was approved by the University of Medicine and Pharmacy \"Grigore T. Popa\" Ethics Committee, approval number 57/17.03.2021. Informed Consent Statement: Informed consent was obtained from all subjects involved in this study. ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare no conflicts of interest. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.3390/molecules191015611	Anti-Fibrosis Effect of Scutellarin via Inhibition of Endothelial–Mesenchymal Transition on Isoprenaline-Induced Myocardial Fibrosis in Rats	<jats:p>Scutellarin (SCU) is the major active component of breviscapine and has been reported to be capable of decreasing myocardial fibrosis. The aim of the present study is to investigate whether SCU treatment attenuates isoprenaline-induced myocardial fibrosis and the mechanisms of its action. Rats were injected subcutaneously with isoprenaline (Iso) to induce myocardial fibrosis and rats in the SCU treatment groups were intraperitoneally infused with SCU (10 mg·kg−1·d−1 or 20 mg·kg−1·d−1, for 14 days). Post-treatment, cardiac functional measurements and the left and right ventricular weight indices (LVWI and RVWI, respectively) were analysed. Pathological alteration, expression of type I and III collagen, Von Willebrand factor, α-smooth muscle actin, cluster of differentiation-31 (CD31), and the Notch signalling proteins (Notch1, Jagged1 and Hes1) were examined. The administration of SCU resulted in a significant improvement in cardiac function and decrease in the cardiac weight indices; reduced fibrous tissue proliferation; reduced levels of type I and III collagen; increased microvascular density; and decreased expression of α-smooth muscle actin and increased expression of CD31, Notch1, Jagged1 and Hes1 in isoprenaline-induced myocardial fibrosis in rats. Our results suggest that SCU prevents isoprenaline-induced myocardial fibrosis via inhibition of cardiac endothelial-mesenchymal transition potentially, which may be associated with the Notch pathway.</jats:p>	[ { "section_content": "Myocardial fibrosis plays a major role in the occurrence and development of a number of different heart diseases.Studies have shown that the occurrence of chronic heart failure is closely related with myocardial fibrosis, with this fibrosis leading to irreversible heart damage and thereby altering heart function from compensated to decompensated.Thus, prevention of myocardial fibrosis could theoretically improve cardiac function and even reverse ventricular remodelling. EndoMT is a complex biological processin by which endothelial cells lose their specific markers and acquire a mesenchymal or myofibroblastic phenotype and express mesenchymal cell products such as α smooth muscle actin (α-SMA) and type I collagen [1].The effects of EndMT in embryonic development, tissue regeneration, wound healing, and neoplasia have been thoroughly investigated, and, recently, EndMT has also been reported to play a role in fibrosis in various tissues, including the lungs, kidneys and the mesenteric and coronary arteries.Zeisberg et al. recently reported on EndMT in heart fibrosis [2], which has opened a new chapter of myocardial fibrosis mechanism research. Notch is a highly conserved signalling pathway between adjacent cells that mediates key cell fate decisions during proliferation, differentiation, and apoptosis.Aberrant Notch signalling can cause abnormal heart development, and recent studies have shown that Notch signalling plays an important role in anti-fibrosis treatment.In transgenic mice overexpressing the Notch ligand Jagged1, Notch was found to inhibit the development of cardiomyocyte hypertrophy and transforming growth factor-β/connective tissue growth factor-mediated cardiac fibrosis [3]. Scutellarin (SCU, 5,6,4-trihydroxyflavone-7-O-glucoronide, Figure 1) is a flavone and the major active component of breviscapine.SCU is a herbal medicine widely used for the treatment of cerebrovascular diseases and has been demonstrated to have anti-apoptotic, antioxidant, anti-inflammatory, and calcium channel antagonist properties in the researches of nervous system disease.The studies related to the physicochemical property and bioavailability of SCU also have been done to ensure the optimal utilization of SCU [4,5].Recently, SCU is found to display beneficial cardiovascular effects, including vasorelaxant effects [6], inhibition of high glucose-mediated vascular inflammation [7], angiogenesis-promoting effects [8], and alleviation of cardiac dysfunction and interstitial fibrosis [9].However, the specific mechanism behind anti-fibrosis effect is still unclear.On the basis of these observations, the aims of the present study were to investigate whether SCU treatment reduces isoprenaline (Iso)-induced myocardial fibrosis in a rat model, to examine the effects of SCU on EndMT and the expression of Notch signalling proteins, and to investigate the relationship between these changes and myocardial fibrosis. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "", "section_name": "Results and Discussion", "section_num": "2." }, { "section_content": "Iso was found to induce significant decreases in LVSP, +dp/dtmax and -dp/dtmax, and an increase in LVEDP (p < 0.01), whereas treatment with SCU attenuated these Iso-induced changes in ventricular function (p < 0.05; Table 1). ", "section_name": "SCU Improves Cardiac Function", "section_num": "2.1." }, { "section_content": "LVWI and RVWI were significantly higher in the Iso-treated group than in the control group (p < 0.01).Treatment with both high-dose and low-dose SCU resulted in significant decreases in LVWI and RVWI compared to the Iso-treated group (p < 0.01).However, no significant differences were observed between the two SCU-treated groups (Table 2). ", "section_name": "SCU Treatment Results in Decreased LVWI and RVWI", "section_num": "2.2." }, { "section_content": "Heart tissues from Iso-treated rats showed widespread fibrous tissue proliferation, myocardial structure disorder, myocardial hypertrophy, vacuolization and leukocyte infiltration compared with the control group.Treatment with SCU produced a marked improvement in Iso-induced fibrous tissue proliferation, subendocardial necrosis, and leukocyte infiltration (Figure 2A).Cells were immunostained with antibodies against α-sma (myofibroblastic phenotype; green) and CD31 (endothelial phenotype; red), and the nuclei were labelled using DAPI dihydrochloride (blue).Magnification ×200.* p < 0.05, ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. Iso group. ", "section_name": "Histopathological Observations of the Myocardium", "section_num": "2.3." }, { "section_content": "Stained myocardium from Iso-treated rats showed widespread blue fibrous tissue (p < 0.01, Figure 2B,D) and significantly increased concentrations of type I and type III collagen in the tissue homogenate compared with the control group (p < 0.01, Table 3).Treatment with both high-dose and low-dose SCU significantly decreased Iso-induced fibrous tissue proliferation and concentrations of type I and type III collagen (p < 0.01 for all) (Figure 2B,D, Table 3). ", "section_name": "SCU Treatment Results in Decreased Myocardial Fibrosis", "section_num": "2.4." }, { "section_content": "The MVD in the Iso-treated group was significantly lower than in the control group (p < 0.01).Treatment with high-dose SCU resulted in a significant increase of MVD compared with the Iso-treated group (p < 0.01).Interestingly, the MVD in the high-dose SCU group was even higher than in the control group (p < 0.05).Low-dose SCU treatment also resulted in an obvious increase in MVD compared with the Iso-treated group (p < 0.01), but this was not as significant as for the high-dose group (Figure 2C,E). ", "section_name": "SCU Treatment Increases the Microvascular Density (MVD)", "section_num": "2.5." }, { "section_content": "Vascular endothelial cells from Iso-treated rats showed an increase in the intensity of α-sma fluorescence and a decrease in the intensity of CD31 fluorescence compared with the control group (p < 0.01).Treatment with SCU significantly attenuated the Iso-induced changes in intensity of α-sma and CD31 fluorescence (p < 0.05; Figure 2F,G). ", "section_name": "Immunofluorescence Observations of the Myocardium", "section_num": "2.6." }, { "section_content": "The expression of CD31 protein and α-sma protein, as determined by western blotting was significantly lower and higher in the Iso-treated group than in the control group respectively (p < 0.01; Figure 3).Treatment with SCU resulted in an increase in the expression of CD31 protein and a decrease in the expression of α-sma protein compared with the Iso-treated group (p < 0.05; Figure 3).Interestingly, the expression of CD31 protein in the high-dose SCU group was even higher than in the control group (p < 0.01; Figure 3A).(C) The expression of Hes1 protein.Notch 1, Jagged1 and Hes1 protein expression levels were significantly lower in the Iso-treated group than in the control group.Treatment with high-dose SCU resulted in significant increases of these proteins compared with both the Iso-treated and control groups.Low-dose SCU treatment also resulted in an obvious increase in these proteins compared with the Iso-treated group, but was not as significant as for high-dose SCU treatment. ", "section_name": "SCU Treatment Leads to an Increase in the Expression of CD31 Protein and a Decrease in the Expression of α-sma Protein in Isoprenaline-Induced Myocardial Fibrosis in Rats", "section_num": "2.7." }, { "section_content": "Jagged1, Notch 1 and Hes1 protein expression levels, as determined by western blot analyses, were significantly lower in the Iso-treated group than in the control group (p < 0.01).Treatment with high-dose SCU resulted in significant increases of these proteins compared with both the Iso-treated and control groups (p < 0.01 for both).Moreover, a significant increase in these proteins was observed after low-dose SCU treatment compared with the Iso-treated group (p < 0.05), but this was not as significant as for the high-dose group (Figure 4). ", "section_name": "SCU Increases the Expression of Jagged1, Notch 1, and Hes1 Proteins", "section_num": "2.8." }, { "section_content": "In our study, we found the Iso-treated rats showed lower cardiac function, higher LVWI and RVWI, impaired cardiac structure, increased fibrous tissue proliferation and levels of type I and III collagen compared with the control group, and the administration of SCU alleviated these changes.Our results demonstrate the anti-fibrosis effect of SCU in isoprenaline-induced myocardial fibrosis in rats.The improvement of myocardial fibrosis was more obvious in rats administrated with high-dose SCU (20 mg/kg/d) than those received 10 mg/kg/d, suggesting the anti-fibrosis effect of SCU may be dose related. In recent years, numerous studies have shown that EndMT plays an important role in fibrosis.Zeisberg et al. recently reported on EndMT in heart fibrosis [1].After mice underwent aortic banding to induce myocardial fibrosis, they found specific labeled endothelial cells scattered throughout the fibrotic area expressed multiple myofibroblastic phenotypes.In vitro, acquisition of a spindle-shaped morphology upon transforming growth factor-β1 (TGF-β1), which is known to promote and induce EndMT [10,11], endothelial cells also gained a fibroblast-like phenotype, whereas untreated control cells preserved their endothelial cell shape and phenotype.During EndMT, the biological function of edothelial cells also get close to fibroblasts. EndMT is a complex biological process that endothelial cells lose their endothelial features and gain mesenchymal or myofibro-blastic properties.Goumans et al. recently pointed out that in cardiac fibrosis, EndoMT represents the most important contributor to the generation of fibrotic tissue [12]. On the one hand, when endothelial cells are damaged, they secrete increased amounts of TGF-β, angiotensin-II, and endothelin-1, which are known to induce myocardial fibrosis, whereas the secretion of nitric oxide and bradykinin, which are known to suppress fibrosis, is decreased.Meanwhile, loss of microvascularity is also known to be involved in the process of tissue fibrosis [13].However, EndMT is associated with endothelial cell damage and decreases in microvessel density. On the other hand, in the heart, as well as in these other organs, the predominant cellular mediators of fibrosis are thought to be (myo)fibroblasts [12].Myofibroblasts, the activated form of cardiac fibroblasts, are capable of secreting extracellular matrix components such as collagen, fibronectin, and laminin to promote the development of fibrosis and about 27%~35% of myofibroblasts were of endothelial origin through EndMT [2]. In the present study, we found that vascular endothelial cells from Iso-treated rats showed decreased CD31 protein and MVD, and increased expression of type I/III collagen and α-sma compared with control rats, which indicates attenuated endothelial characteristic and enhanced fibroblast property, suggesting the EndMT in Iso-induced myocardial fibrosis.While treatment with SCU attenuated these effects, suggesting that SCU treatment improves myocardial fibrosis via inhibition of EndMT potentially.Interestingly, the expression of CD31 protein and MVD in SCU groups was increased, and in the high-dose SCU group the expression of CD31 protein and MVD was even higher than in the control group.Thus, we speculate that SCU might have angiogenesis-promoting effect in intervening in myocardial fibrosis.And, the angiogenesis-promoting effect of SCU has been demonstrated recently by Ma et al., and this effect may be dose related [8].Indeed, further studies should be done in the future to confirm the angiogenesis-promoting effect of SCU in intervening in myocardial fibrosis and investigate the mechanisms of its action, which may provide a new scope for the treatment of cardiac fibrosis. The Notch signalling pathway involves the Notch-1/2/3/4 receptors and their transmembrane ligands (Jagged-1/2 and agged-1/3/4).Upon ligand binding, the Notch intracellular domain is released by the γ-secretase complex, migrates into the nucleus, and interacts with transcriptional repressors, thereby regulating target genes such as the basic helix-loop-helix proteins Hes and Hey. A number of previous studies have shown that the Notch signalling pathway regulates myocardial fibrosis.In transgenic mice overexpressing the Notch ligand Jagged1, Notch was found to inhibit the development of cardiomyocyte hypertrophy and TGF-β/connective tissue growth factor-mediated cardiac fibrosis [3].Moreover, in vitro studies have demonstrated that Notch signalling influences the differentiation of fibroblasts into myofibroblasts [14,15]. In this study, the protein expression of Notch 1, Jagged1, and Hes1 proteins in the Iso-treated group were significantly lower than in the control group, and treatment with SCU was found to increase the expression of these proteins to the point when, in the high-dose SCU group, it was even higher than in the control group.These results suggest that suppression of myocardial fibrosis by SCU may be associated with the Notch pathway.However, the evidence is limited and further study should be done. In myocardial fibrosis, endothelial cells provide sources for fibroblasts via EndMT.TGF-β is often used to to promote and induce EndMT, and Notch signalling has been previously demonstrated to inhibit TGF-β-induced EndMT and myocardial fibrosis [3].Furthermore, Previous research found that TGF-β1 could increase the expression of α-sma and down-regulate Notch receptors 1, 3, and 4 in cardiac fibroblasts, and that Notch signalling inhibitors increased the expression of α-sma [15].In this study, the Iso-treated rats showed increased expression of α-sma and decreased expression of CD31, Notch1, Jagged1 and Hes1 compared with control rats, while the administration of SCU resulted in decreased expression of α-sma and increased expression of CD31, Notch1, Jagged1 and Hes1, that was, SCU restrained EndMT and up-regulated of Notch signalling in our rat model.According to this, we made a hypothesis that the probable molecular mechanism of SCU inhibiting the EndMT in myocardial fibrosis may be associated with the Notch pathway.And further studies should assess how Notch pathway proteins are interlinked and cooperated to mediate the effects of SCU on EndMT. ", "section_name": "Discussion", "section_num": "2.9." }, { "section_content": "", "section_name": "Experimental Section", "section_num": "3." }, { "section_content": "SCU (Shanghai Boyun Biotech, Shanghai, China, 200 mg) was diluted with water for injection (100 mL) and the pH value was adjusted with weak alkaline solutions such as sodium bicarbonate and sodium hydroxide until completely dissolved.SCU solution is not very stable [4], thus, the solutions should be used immediately after preparation, or stored at -20 °C. ", "section_name": "SCU Preparation Method", "section_num": "3.1." }, { "section_content": "Forty male SD rats (weight, 200-220 g; age, approximately 6 weeks) were supplied by Wenzhou Medical University Laboratory Animal Centre (Wenzhou City, Zhejiang Province, China).The animals were housed individually in cages under hygienic conditions and placed in a controlled environment with a 12 h (light)-12 h (dark) cycle at 22 ± 3 °C and 45% ± 10% humidity for 7 days before the initiation of the experiments.The animals were allowed a standard commercial diet (Laboratory Animal Centre of Zhejiang Province, Hangzhou, China) and tap water.The study was approved by the institutional research ethics committee of Wenzhou Medical University (protocol # wydw2013-0054). At the start of the study, the rats were randomly divided into four groups (10 animals in each group): (1) myocardial fibrosis model (Iso) group; (2) low-dose SCU group (10 mg/kg/d); (3) high-dose SCU group (20 mg/kg/d); and (4) control group.On day 1-7, rats were subcutaneously injected with Iso (5 mg/kg body mass -1 , Sigma, St. Louis, MO, USA) to induce experimental myocardial fibrosis according to the related literature [16], while rats in the control group were subcutaneously injected with normal saline.Subsequently, from day 2 to day 15, rats in the low-dose and high-dose SCU groups were intraperitoneally infused with SCU 10 mg/kg/d and 20 mg/kg/d, respectively.The method of administration and dosage were referenced to previous researches [8,17]. ", "section_name": "Animals and Treatments", "section_num": "3.2." }, { "section_content": "", "section_name": "Methods", "section_num": "3.3." }, { "section_content": "The rats were anaesthetised using 1% pentobarbital sodium (40 mg•kg -1 ), and lack of limbs pinch reflex indicated the surgical anaesthesia sufficient for operation.The right carotid artery was separated to allow insertion of a catheter containing 0.05% heparin saline to the left ventricle, and the other end of the catheter was connected to a PowerLab polygraph recorder (AD Instruments, Castle Hill, Australia) to record the changes of the left ventricular mean systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and the maximum rate of change in left ventricular pressure (+dp/dtmax, -dp/dtmax). ", "section_name": "Cardiac Functional Measurements", "section_num": "3.3.1." }, { "section_content": "After being humanely euthanized by bleeding, precooled saline (4 °C) was infused into the left ventricle of the rats until the heart and kidney paled.The heart was rapidly excised and rinsed in cold normal saline.Subsequently, the left and right ventricles were separated and weighed, and the left and right ventricular weight indices (LVWI and RVWI) were calculated as the left and right ventricular free wall mass (mg) divided by body mass (g), respectively. ", "section_name": "Left Ventricular Weight Index (LVWI) and Right Ventricular Weight Index (RVWI)", "section_num": "3.3.2." }, { "section_content": "Cardiac apex samples of the left ventricle were fixed in 4% buffered paraformaldehyde solution and embedded in paraffin.Paraffin sections (4-μm thick) were stained with haematoxylin and eosin (Beyotime Institute of Biotechnology, Shanghai, China).The sections were examined by light microscopy (Nikon Corporation, Tokyo, Japan) and photomicrographs were taken at ×200 magnification. ", "section_name": "Haematoxylin and Eosin Staining of the Left Ventricular Myocardium", "section_num": "3.3.3." }, { "section_content": "Paraffin sections were stained with Masson trichrome (GenMed Scientifics Inc., Boston, MA, USA), resulting in cardiomyocytes staining red and fibrous tissue staining blue.The sections were examined using light microscopy, photographs were taken at the magnification of ×40 and ×200.Five non-repeating visual fields, magnification ×200, were randomly selected, myocardial collagen areas were measured using Image-Pro Plus (Media Cybernetics, Inc., Bethesda, MD, USA) and the areas were averaged. ", "section_name": "Masson Trichrome Staining of the Left Ventricular Myocardium", "section_num": "3.3.4." }, { "section_content": "A piece of the left ventricular myocardium (100 mg) was cut into smaller pieces, added to 1 mL phosphate buffered saline (PBS) (pH 7.4), and vortexed on ice.After centrifugation at 3000 rpm for 20 min, the supernatant was separated and the concentrations of type I and III collagen were measured using ELISA (Shanghai Boyun Biotech) according to the manufacturer's protocol. ", "section_name": "Enzyme-Linked Immunoassay (ELISA) for Type I and III Collagen", "section_num": "3.3.5." }, { "section_content": "Von Willebrand factor was used as a marker of vascular endothelial cells to indicate blood vessels.Endogenous peroxidase activity was blocked with 3% methanol-H2O2.Nonspecific sites were blocked with 5%-10% goat serum before incubation with the primary antibody (Anti-Von Willebrand factor antibody, Abcam, Cambridge, UK, 1:600) in 0.1 M PBS overnight at 4 °C.After washing, reagents 1 and 2 of the Polymer HRP Detection System for rabbit primary antibodies (Zhongshan Jinqiao, Beijing, China) were added and washed sequentially, and developed with horseradish peroxidase and diaminobenzidine chromogen (Zhongshan Jinqiao).The blood vessels were observed at ×100 and ×400 magnification before being imaged and counted at ×200 magnification.Stained single cells or cell clusters clearly separated from the surrounding vessels, cardiomyocytes, and other tissues were regarded as microvessels.The number of these microvessels was calculated as the average microvascular density (MVD) in five randomly selected non-repeating visual fields. ", "section_name": "Immunohistochemistry", "section_num": "3.3.6." }, { "section_content": "CD31 is a member of the immunoglobulin superfamily, makes up a large portion of endothelial cell intercellular junctions and is likely involved in leukocyte transmigration, angiogenesis, and integrin activation.Here, a anti-CD31 antibody was used to label endothelial cells.And, α-SMA is the major morphological characteristic of myofibroblasts.For IFA, endogenous peroxidase activity was blocked with 3% methanol-H2O2, and the nonspecific sites were treated with 10% foetal bovine serum.A mix of primary antibodies (anti-α-smooth muscle actin [sma] antibody, Wuhanboshide, Wuhan, China, 1:100; anti-cluster of differentiation [CD]31 antibody, Santa Cruz Biotechnology, Inc., SantaCruz, CA, USA, 1:30) was added to the sections and incubated overnight at 4 °C.After washing, a mix of secondary antibodies (DyLight 488 AffiniPure Goat Anti-Mouse IgG(H+L) and DyLight 594 AffiniPure Goat Anti-Rabbit IgG(H+L), EarthOX, San Francisco, CA, USA, 1:300) and DAPI staining solution (Beijing Leagene Biotechnology, Beijing, China) were added sequentially, followed by washing.Photographs were taken at ×200 magnification with a fluorescence microscope (Nikon Corporation, Tokyo, Japan) and processed with Image-Pro Plus.Cells were immunostained with antibodies against α-sma (myofibroblastic phenotype; green) and CD31 (endothelial phenotype; red), and the nuclei were labelled by DAPI dihydrochloride (blue). ", "section_name": "Immunofluorescence Methods (IFA)", "section_num": "3.3.7." }, { "section_content": "Equal amounts of the samples (50 µg) were loaded on gels for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology).The membranes were blocked with 5% skim milk and incubated with the primary antibodies (anti-CD31 antibody, Santa Cruz Biotechnology, Inc., 1:1,000; anti-α-smooth muscle actin [sma] antibody, Wuhanboshide, China, 1:1000; anti-notch1 antibody, Cst, Danvers, MA, USA, 1:1000; anti-Jagged1 antibody, Cst, USA, 1:1000; anti-Hes1 antibody, Abcam, Cambridge, UK) overnight at 4 °C.Next, the membranes were incubated with the secondary antibody (HRP-conjugated goat anti-rabbit IgG(H+L), Bioworld Technology, Nanjing, China, 1:5000-1:20,000) for 2 h at room temperature.Immunoreactive bands were detected using Chemiluminescent HRP Substrate (Applygen Technologies, Beijing, China), and scans were obtained using the Bio-Rad gel image analysis system (BioRad, Hercules, CA, USA) and processed using Image-Pro Plus (Media Cybernetics, Inc.).The housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Bioworld Technology, Nanjing, China, 1:5000) was used as the loading control. ", "section_name": "Western Blot", "section_num": "3.3.8." }, { "section_content": "All results are expressed as mean ± standard error (SEM).All statistical analyses were performed with SPSS software (version 16.0; SPSS Inc., Chicago, IL, USA) by Student's t test, and one-way ANOVA with the next modified t-test.p values < 0.05 were considered statistically significant.The low number of animals and the many different statistical models may increase the risk of statistical errors. ", "section_name": "Statistical Analysis", "section_num": "3.3.9." }, { "section_content": "Our results suggest that SCU prevents isoprenaline-induced myocardial fibrosis via inhibition of cardiac endothelial-mesenchymal transition potentially, which may be associated with the Notch pathway. ", "section_name": "Conclusions", "section_num": "4." } ]	[ { "section_content": "This research was supported by the Zhejiang Provincial Natural Science Foundation of China (LY12H02004), and in part by the Foundation for the Program of Science and Technology Department of Zhejiang Province of China (2014C33166) and the Foundation for the Program of the Provincial Health Department of Zhejiang Province of China (2014KYA136). ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/10/15611/s1. Hao Zhou conceived of the study, drafted the design, carried out experimental procedures and drafted the manuscript; Jie-Jie Cai carried some of the experimental procedures, participated in its design and helped to draft the manuscript; Xi Zhou participated in the design of the study; Xiao Chen, Lingzhi Chen and Gaoshu Zheng, participated in some experimental procedures; Weijian Huang and Huaiqin Zhang participated in the design of the study and helped to draft the manuscript.All authors read and approved the final manuscript. The authors declare no conflict of interests. ", "section_name": "Supplementary Materials", "section_num": null }, { "section_content": "Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/10/15611/s1. ", "section_name": "Supplementary Materials", "section_num": null }, { "section_content": "Hao Zhou conceived of the study, drafted the design, carried out experimental procedures and drafted the manuscript; Jie-Jie Cai carried some of the experimental procedures, participated in its design and helped to draft the manuscript; Xi Zhou participated in the design of the study; Xiao Chen, Lingzhi Chen and Gaoshu Zheng, participated in some experimental procedures; Weijian Huang and Huaiqin Zhang participated in the design of the study and helped to draft the manuscript.All authors read and approved the final manuscript. ", "section_name": "Author Contributions", "section_num": null }, { "section_content": "The authors declare no conflict of interests. ", "section_name": "Conflicts of Interest", "section_num": null } ]
10.1038/cddis.2015.415	Genetic profiling of CLL: a ‘TP53 addict’ perspective	Chronic lymphocytic leukaemia (CLL) is a B-cell malignancy with a highly variable clinical course. Whereas some patients require treatment relatively soon after diagnosis, others may stay free of symptoms for many years with a standard ‘watch and wait’ surveillance approach. Genomic analysis of asymptomatic patients and patients with progressive or relapsed refractory disease makes CLL an excellent model to study the course of genetic modifications in relation to the pathobiology of this disease.	[ { "section_content": "Chronic lymphocytic leukaemia (CLL) is a B-cell malignancy with a highly variable clinical course.Whereas some patients require treatment relatively soon after diagnosis, others may stay free of symptoms for many years with a standard 'watch and wait' surveillance approach.Genomic analysis of asymptomatic patients and patients with progressive or relapsed refractory disease makes CLL an excellent model to study the course of genetic modifications in relation to the pathobiology of this disease. The use of karyotype analysis first demonstrated that the deletions of chromosomes 13q, 17p, and 11q as well as trisomy 12 were recurrent aberrations in CLL.Mutations in several cancer genes were subsequently identified in these regions: ATM and BIRC3 in 11q or TP53 in 17p.Sanger sequencing as well as fluorescence in situ hybridisation or genomic arrays have further identified a wide spectrum of genomic modifications emphasising the marked genetic heterogeneity of CLL. Considerable progress has been made in the field of CLL genetics over last 3 years, with the publication of multiple studies using next generation sequencing (NGS) culminating with two recent reports in Nature, which describe the whole exome/genome sequencing of 990 CLL patients. 1,2Both studies confirmed the considerable genetic heterogeneity of CLL as well as its evolution during disease progression. Tumour genome sequencing shatters genetic information into small pieces, which must be then reconstructed like a giant jigsaw puzzle without a picture on the box.Assembling genetic alterations, based on previous knowledge about biological processes, allows investigation of entire sets of mutations in a tumour and determination of the targeted pathways.The fundamental goal is to decrease the data set concerning countless altered genes and proteins to a smaller and more meaningful set of altered pathways.This strategy will generate testable hypotheses, identify tumour subtypes with clinically distinct outcomes, characterise the cancerspecific and cross-cancer pathways and help to identify drug targets.Furthermore, analysis of large cohorts of patients has revealed that the driver mutations targeting multiple components of a single pathway are mutually exclusive on tumour samples.Identification of this type of exclusive pattern is important for the understanding of cancer progression, leading to a stimulating feedback between the patient and the lab, as this analysis may lead to the proposal of genes for targeted therapy. The TP53 network is a paradigm for this type of analysis, as it is impaired in most human cancers. 3The backbone of this pathway is the TP53 autoregulatory feedback loop and its negative regulator MDM2 (Figure 1).Depending on the type of stress, multiple upstream signals can disrupt this regulation leading to TP53 activation and initiation of a complex transcriptional program, which is essential to maintain cellular homoeostasis. Inactivation of several members of this network in CLL has already been clearly established with an apparent focus on the DNA-damage pathway with ATM and POT1 mutations (Figure 1).Although the mutual exclusivity of ATM and TP53 alterations has already been reported, the observation that, in the series of 58 POT1 mutations reported by Puente et al. and Landau et al., only one patient also harboured a TP53 mutation is a strong argument to include POT1 alterations in the TP53 network targeted in CLL.POT1 is an essential component of shelterin, a protein complex that shapes and safeguards human telomeres and activates the TP53 pathway via ATR kinase, inducing telomere shortening or uncapping, and therefore, preventing chromosomal instability.Whether or not these tumours exhibit a particular genetic instability is currently unknown.The link between POT1 and TP53 is reinforced by the recent finding of POT1 germline mutations in three TP53-negative Li-Fraumeni-like families with cardiac angiosarcoma, a very rare malignant tumour. 4A few ATR mutations have also been described in CLL, but it is unclear whether they are mutually exclusive to POT1 (and ATM) mutations. RPS15 mutations shed light on another aspect of the TP53 pathway (Figure 1).Accurate ribosome biogenesis is carefully controlled to prevent quantitative and qualitative protein translation. 5The MDM2 protein is critical for this nucleolar response via binding of 5S RNP, which contains 5SRNA, RPL11 and RPL5 in response to impaired ribosomal biogenesis. 6More recently, other proteins associated with the small subunit of the ribosome (RPS15 or RPS30) have been shown to bind and inactivate MDM2, leading to a strong TP53 response and cell death. 7It has been hypothesised that RPS15 (like several other ribosomal proteins) could act as a 'detector' of impaired ribosomal explaining why RPS15 mutations can contribute to tumourigenicity.Although only a small number of patients harbour RPS15 mutations, these mutations tend to be exclusive of TP53 alterations and are associated with shorter progression-free survival (PFS). In a seminal paper, Rossi et al. 8 demonstrated BIRC3 inactivation in a subset of patients who were refractory to fludarabine therapy.This observation was confirmed by the work of Landau et al. and Puente et al., who both showed that BIRC3 mutations are associated with shorter survival, a feature shared by patients with ATM or TP53 alterations.A remarkable feature of BIRC3 mutations is their occurrence in tumours not presenting any TP53 mutations, suggesting that they are associated with a common pathway.BIRC3, also known as cIAP2 (cellular inhibitor of apoptosis proteins), is a regulator of canonical NF-kB signalling downstream from the TNF-R1 receptor, and also functions as a negative regulator of the non-canonical NF-kB pathway via RING finger domain-dependent ubiquitination of NIK.In a cellular model, downregulation of BIRC3/cIAP2 led to TP53 degradation via NF-KB-dependent phosphorylation and activation of mdm2. 9On the other hand, most BIRC3 mutations are localised in the carboxy terminus, resulting in proteins that are deficient for their ubiquitination activity, suggesting a possible gain of function. Despite the multiple links between the TP53 and NF-kB pathways, the mutually exclusive nature of BIRC3 and TP53 mutations cannot be easily explained, but should be explored in more detail to gain insight into the mechanisms, leading to resistance to therapy. MicroRNAs are an important component of the BCR (B-cell receptor) signalling pathway.The signature profile of microRNAs can distinguish normal B cells from malignant CLL.Several microRNAs regulated by TP53, such as miR-15a, miR-161 localised on chromosome 13 or mi-R34A/b localised on chromosome 11, are frequently deregulated in CLL.Whether or not, these defects impair the TP53 pathway currently remains unknown, but should be investigated to gain more insight into the role of microRNA defects. Finally, a few words on TP53 mutations.Although seemingly infrequent in the early phase of the disease, TP53 mutations are often found in patients with relapse/refractory disease and poor survival. 10Nonetheless, deep NGS has demonstrated the presence of very small subclones in upto 9% of cases at diagnosis, and retrospectively in early samples of patients who exhibited larger clones at later stages of progression. 11It is noteworthy that approximately one half of these patients did not harbour a 17p deletion, and therefore, retained the wildtype TP53 allele.It is currently unknown whether TP53 mutations observed in patients with and without 17p deletion have different biochemical properties.The importance of TP53 during disease progression is also emphasised by the identification of multiple subclones harbouring different TP53 mutations in leukaemic cells.The presence of TP53 mutation has been linked to chemorefractoriness, and expansion of TP53-mutated subclones has been clearly demonstrated in patients relapsing after initial chemotherapy. Defining the origin of these clones, how they evolve and perhaps compete during tumour progression, are among the several questions that NGS will be able to resolve in the near future. The presence of TP53 mutations represent an adverse prognostic parameter at all time points of CLL history: at diagnosis, at the time of initial therapy and at relapse.New agents such as BCR inhibitors have provided very promising results, and have been approved by both FDA and EMA for use as first-line therapy in the presence of TP53 alteration.It is therefore highly recommended to test patients for the presence of 17p deletion and TP53 mutation before treatment decisions, even beyond the context of clinical trials. Clearly, several upstream pathways converging to TP53 are impaired in CLL, as well as alteration of the TP53 gene itself.Whether or not this can be considered as a single clinical entity, has to be resolved yet.The observation that ATM, RPS15, TP53 or BIRC3 alterations are associated with shorter overall survival and PFS is strong argument in favour of this hypothesis, which is reinforced by the observation that TP53 mutations are strongly exclusive with BIRC3, ATM and RPS15 mutations in the series of 990 cases of CLL published by Landau et al. and Puente et al.Although the exclusive nature with ATM mutations is not always observed, the discovery that CLL, and more particularly cases of CLL in progression or relapse, display high levels of subclones that could harbour different genotypes could explain this apparent discrepancy.Analysis of clonal evolution as well as single-cell sequencing will be able to refine our knowledge on the genetics of CLL. ", "section_name": "", "section_num": "" } ]	[ { "section_content": "The authors declare no conflict of interest. Figure 1 The TP53 pathway in CLL.The level of TP53 protein is downregulated via binding of proteins, such as, MDM2 that promote TP53 degradation via the ubiquitin/proteasome pathway.As MDM2 is upregulated by TP53, this leads to a regulation loop, which maintains a very low level of TP53 protein in normal cells.This function also requires MDMX, which binds both MDM2 and TP53.Disruption of this equilibrium leading to TP53 accumulation is the main outcome of the upstream pathways to promote a TP53 biological response.In CLL, two pathways associated with DNA damage (double-strand breaks or telomere uncapping) are clearly impaired.Genes depicted in red are impaired in CLL.Whether or not trisomy 12 can alter the TP53 pathway by increasing mdm2 activity is controversial.The links between BIRC3 and the TP53 pathway remain elusive ", "section_name": "Conflict of Interest", "section_num": null } ]
10.1038/ncomms14175	Genome-wide association analysis implicates dysregulation of immunity genes in chronic lymphocytic leukaemia	<jats:title>Abstract</jats:title><jats:p>Several chronic lymphocytic leukaemia (CLL) susceptibility loci have been reported; however, much of the heritable risk remains unidentified. Here we perform a meta-analysis of six genome-wide association studies, imputed using a merged reference panel of 1,000 Genomes and UK10K data, totalling 6,200 cases and 17,598 controls after replication. We identify nine risk loci at 1p36.11 (rs34676223,<jats:italic>P</jats:italic>=5.04 × 10<jats:sup>−13</jats:sup>), 1q42.13 (rs41271473,<jats:italic>P</jats:italic>=1.06 × 10<jats:sup>−10</jats:sup>), 4q24 (rs71597109,<jats:italic>P</jats:italic>=1.37 × 10<jats:sup>−10</jats:sup>), 4q35.1 (rs57214277,<jats:italic>P</jats:italic>=3.69 × 10<jats:sup>−8</jats:sup>), 6p21.31 (rs3800461,<jats:italic>P</jats:italic>=1.97 × 10<jats:sup>−8</jats:sup>), 11q23.2 (rs61904987,<jats:italic>P</jats:italic>=2.64 × 10<jats:sup>−11</jats:sup>), 18q21.1 (rs1036935,<jats:italic>P</jats:italic>=3.27 × 10<jats:sup>−8</jats:sup>), 19p13.3 (rs7254272,<jats:italic>P</jats:italic>=4.67 × 10<jats:sup>−8</jats:sup>) and 22q13.33 (rs140522,<jats:italic>P</jats:italic>=2.70 × 10<jats:sup>−9</jats:sup>). These new and established risk loci map to areas of active chromatin and show an over-representation of transcription factor binding for the key determinants of B-cell development and immune response.</jats:p>	[ { "section_content": "hronic lymphocytic leukaemia (CLL) is an indolent B-cell malignancy that has a strong genetic component, as evidenced by the eightfold increased risk seen in relatives of CLL patients 1 .Our understanding of CLL genetics has been transformed by genome-wide association studies (GWAS) that have identified risk alleles for CLL [2][3][4][5][6][7][8][9] .So far, common genetic variation at 33 loci has been shown to influence CLL risk.Although projections indicate that additional risk variants for CLL can be discovered by GWAS, the statistical power of the individual existing studies is limited. To gain a more comprehensive insight into CLL predisposition, we analysed genome-wide association data from populations of European ancestry from Europe, North America and Australia, identifying nine new risk loci.Our findings provide additional insights into the genetic and biological basis of CLL risk. ", "section_name": "C", "section_num": null }, { "section_content": "Association analysis.After quality control, the six GWAS provided single-nucleotide polymorphism (SNP) genotypes on 4,478 cases and 13,213 controls (Supplementary Tables 1 and2).To increase genomic resolution, we imputed 410 million SNPs using the 1000 Genomes Project 10 combined with UK10K 11 as reference.Quantile-Quantile (Q-Q) plots for SNPs with minor allele frequency (MAF) 40.5% post imputation did not show evidence of substantive overdispersion (l between 1.00 and 1.10 across the studies; Supplementary Fig. 1).Meta-analysing the association test results from the six series, we derived joint odds ratios per-allele and 95% confidence intervals under a fixedeffects model for each SNP and associated P values.In this analysis, associations for the established risk loci were consistent in direction and magnitude of effect with previously reported studies (Fig. 1 and Supplementary Table 3). We identified 16 loci where at least one SNP showed evidence of association with CLL (defined as Po1.0 Â 10 À 7 in fixedeffects meta-analysis of the six series) and which were not previously implicated with CLL risk at genome-wide significance (that is, Po5.0 Â 10 À 8 ; Table 1 and Supplementary Tables 4 and5).Where the signal was provided by an imputed SNP, we confirmed the fidelity of imputation by genotyping (Supplementary Table 6).We substantiated the 16 SNPs using de novo genotyping in two studies and in silico replication in two additional studies, totalling 1,722 cases and 4,385 controls.Metaanalysis of the discovery and replication studies revealed genomewide significant associations for eight novel loci (Table 1) at 1p36.11 (rs34676223, P ¼ 5.04 Â 10 À 13 ), 1q42.13 (rs41271473, P ¼ 1.06 Â 10 À 10 ), 4q35.1 (rs57214277, P ¼ 3.69 Â 10 À 8 ), 6p21.31(rs3800461, P ¼ 1.97 Â 10 À 8 ), 11q23.2(rs61904987, P ¼ 2.64 Â 10 À 11 ), 18q21.1 (rs1036935, P ¼ 3.27 Â 10 À 8 ), 19p13.3(rs7254272, P ¼ 4.67 Â 10 À 8 ) and 22q13.33(rs140522, P ¼ 2.70 Â 10 À 9 ).We also confirmed 4q24 (rs71597109, P ¼ 1.37 Â 10 À 10 ), which has previously been identified as a suggestive risk locus 9 .Conditional analysis of GWAS data showed no evidence for additional independent signals at these nine loci.In the remaining seven loci that did not replicate with genome-wide significance, the 9q22.33 locus (rs7026022, P ¼ 7.00 Â 10 À 8 ) remains suggestive (Supplementary Table 5).In analyses limited to the exomes of 141 CLL cases from 66 families, we found no evidence to suggest that any of the association signals might be a consequence of linkage disequilibrium (LD) with a rare disruptive coding variant. Several of the newly identified risk SNPs map in or near to genes with established roles in B-cell biology, hence representing credible candidates for susceptibility to CLL.The 4q24 association marked by rs71597109 (Fig. 2) maps to intron 1 of the gene encoding BANK1 (B-cell scaffold protein with ankyrin repeats 1), a B-cell-specific scaffold protein.SNPs at this locus have been associated with systemic lupus erythematosus risk 12 .BANK1 expression is only seen in functional B-cell antigen receptor (BCR)-expressing B cells, mediating effects through LYN-mediated tyrosine phosphorylation of inositol triphosphate receptors.BANK1-deficient mice display higher levels of mature B cells and spontaneous germinal centre B cells 13 , while studies in humans found lower BANK1 transcript levels in CLL versus normal B cells 14 .The 19p13.3 association marked by rs7254272 (Fig. 2) maps 2.5 kb 5 0 to ZBTB7A (zinc finger and BTB domaincontaining protein 7a, alias LRF, leukaemia/lymphoma-related factor, pokemon).ZBTB7A is a master regulator of B versus T lymphoid fate.Loss of ZBTB7A results in aberrant activation of the NOTCH pathway in lymphoid progenitors.NOTCH is constitutively activated in CLL and is a determinant of resistance to apoptosis in CLL cells.rs34676223 at 1p36.11 maps B10 kb upstream of MDS2 (Fig. 2), which is the fusion partner of ETV6 in t(1;12)(p36;p13) myelodysplasia.Based on RNA sequencing (RNA-seq) data from patients, MDS2 is overexpressed in CLL versus normal cells and also differentially expressed between two experimentally determined CLL subgroups 14 .The SNP rs57214277 maps to 4q35.1 and resides B140 kb centromeric to IRF2 (interferon regulatory factor 2, Fig. 2).Interferon (IFN)ab, a family of antiviral immune genes, induces IRF2 that inhibits the reactivation of murine gamma herpesvirus 15 .Furthermore, SNPs in strong LD with rs57214277 are associated with increased expression of IRF2 as well as trans-regulation of a network of genes in lipopolysaccharide and IFNg-treated monocytes 16 .rs140522 maps to 22q13.33 (Fig. 2), which has previously been associated with multiple sclerosis risk 17 .This region of LD contains four genes, of which only NCAPH2 (non-SMC condensin II complex subunit H2) shows differential expression between CLL and normal B cells 14 (B2.5-foldlower levels in CLL), and plays an essential role in mitotic chromosome assembly and segregation.rs41271473, rs3800461, rs61904987 and rs1036935 mark genes that have roles in WNT signalling (RHOU), autophagy (C6orf106), transcriptional activation (CXXC1), kinetochore association (SKA1, ZW10) and protein degradation (USP28, TMPRSS5; Fig. 3). New CLL risk SNPs and clinical phenotype.We tested for differences in the associations by sex or age at diagnosis for each of the nine risk SNPs using case-only analysis, and observed no relationships (Supplementary Data 1).In addition, case-only analysis in a subset of studies provided no evidence for associations between risk SNP genotypes and IGVH (immunoglobulin variable region heavy chain) mutation subtype (Supplementary Data 1) or overall patient survival (Supplementary Table 7).Collectively, these data suggest that RAF is risk allele frequency across all of the discovery and replication data sets, respectively.ORs are derived with respect to the risk allele.Text in bold highlight the P-value in the combined data. these nine risk variants have generic effects on CLL development rather than tumour progression per se. Functional annotation of new risk loci.To gain insight into the biological basis underlying the novel association signals, we first evaluated profiles for three histone marks (H3K4me1, H3K27ac marking active chromatin and the repressive mark H3K27me3) at each locus, in GM12878 lymphoblastoid cell line (LCL; ref. 18) as well as primary CLL samples 19 (Supplementary Fig. 2).We also examined ATAC-seq profiles from CLL samples and primary B cells as a marker of chromatin accessibility 19,20 .Since the strongest associated GWAS SNP may not represent the causal variant, we examined signals across an interval spanning all variants in LD r 2 40.2 with the sentinel SNP (based on the 1000 Genomes EUR reference panel).These data revealed regions of active chromatin state at all nine risk loci, in at least one of the cell types.Furthermore, based on the analyses of Hnisz et al. 21, five of the loci fall within regions designated as 'super enhancers' in either LCLs or CD19 B cells (Supplementary Fig. 2).Overall, (a-e).Plots (drawn using visPig 62 ) show association results of both genotyped (triangles) and imputed (circles) SNPs in the GWAS samples and recombination rates.À log 10 P values (y axes) of the SNPs are shown according to their chromosomal positions (x axes).The sentinel SNP in each combined analysis is shown as a large circle or triangle and is labelled by its rsID.The colour intensity of each symbol reflects the extent of LD with the top genotyped SNP, white (r 2 ¼ 0) through to dark red (r 2 ¼ 1.0).Genetic recombination rates, estimated using the 1000 Genomes Project samples, are shown with a light blue line.Physical positions are based on NCBI build 37 of the human genome.Also shown are the chromatin-state segmentation track (ChromHMM) for lymphoblastoid cells using data from the HapMap ENCODE Project, and the positions of genes and transcripts mapping to the region of association. these findings suggest that the risk loci annotate regulatory regions and may, therefore, have an impact on CLL risk through modulation of enhancer or promoter activity.Given the possibility that SNPs might influence enhancer or promoter activity by causing changes in transcription factor (TF) binding, we next evaluated the SNPs at each GWAS locus based on their overlap with TF-binding sites.In the absence of comprehensive TF chromatin immunoprecipitation sequencing (ChIP-Seq) data from CLL samples, we used regions of chromatin accessibility defined by ATAC-seq data 19 as a surrogate marker for TF binding, identifying 47 SNPs in LD r 2 40.2 with the sentinel SNPs that also overlapped ATAC-seq peaks.Using motifbreakR 22 to predict whether these SNPs might disrupt TFbinding motifs, we found 478 potentially disrupted motifs, corresponding to 349 TF-binding sites (Supplementary Table 7).Moreover, at 10 of the SNPs, the altered motif matched the TFs bound in ChIP-seq data from the ENCODE project (Supplementary Table 8 and Supplementary Fig. 3).In particular, we noted that rs13149699 at 4q35 (r 2 ¼ 0.83 with lead SNP rs57214277) was predicted to disrupt SPI1 binding.In addition, rs13149699 showed evidence of evolutionary constraint, and in LCL ChIP-seq data, the SNP was bound by SPI1 as well as other TFs with roles in B-cell function including IRF4, PAX5, POU2F2 (alias OCT2) and RELA (Supplementary Table 8). We explored whether there was any association between the genotypes of the nine new risk SNPs and the transcript levels of genes within 1 Mb of each respective variant by performing expression quantitative trait loci (eQTL) analysis using gene expression profiles of 468 CLL cases.In addition, we interrogated publicly accessible expression data on whole blood and LCLs (Supplementary Data 2).There were significant (false discovery rate (FDR)o0.05)and consistent eQTLs between rs3800461 and C6orf106, rs1036935 and SKA1, rs140522 and ODF3B, and rs140522 and TYMP. Biological inference of all CLL risk loci.Given our observation that the nine novel risk loci annotate putative regulatory regions, we sought to examine the epigenetic landscape of CLL risk loci on a broader scale, evaluating the enrichment of both histone a-d).Plots (drawn using visPig 62 ) show association results of both genotyped (triangles) and imputed (circles) SNPs in the GWAS samples and recombination rates.À log 10 P values (y axes) of the SNPs are shown according to their chromosomal positions (x axes).The sentinel SNP in each combined analysis is shown as a large circle or triangle and is labelled by its rsID.The colour intensity of each symbol reflects the extent of LD with the top genotyped SNP, white (r 2 ¼ 0) through dark red (r 2 ¼ 1.0).Genetic recombination rates, estimated using the 1000 Genomes Project samples, are shown with a light blue line.Physical positions are based on NCBI build 37 of the human genome.Also shown are the chromatin-state segmentation track (ChromHMM) for lymphoblastoid cells using data from the HapMap ENCODE Project, and the positions of genes and transcripts mapping to the region of association.modifications (N ¼ 11) and TF binding (N ¼ 82) in GM12878 LCLs, across the new and previously published CLL GWAS risk SNPs.Using the variant set enrichment method of Cowper-Sal lari et al. 23 , we identified regions of strong LD (defined as r 2 40.8 and D 0 40.8) and determined the overlap between these variants and ENCODE ChIP-seq data.Imposing a P value threshold of 5.37 Â 10 À 4 (that is, 0.05/93, based on permutation), we identified a significant enrichment of histone marks associated with active enhancer and promoter elements (HK4Me1, H3K27ac and H3K9ac) as well as actively transcribed regions (H3K36me3).We also identified an over-representation of TF binding for POLR2A, IRF4, RUNX3, NFATC1, STAT5A, PML and WRNIP1 (Fig. 4).In addition, although not statistically significant, POU2F2 showed evidence for enriched binding (P ¼ 7.78 Â 10 À 4 ).Several of these TFs have established roles in B-cell function.OCT2, IRF4 and RUNX3 have been shown to be targeted for hypomethylation in B cells 24 .MYC is a direct target of IRF4 in activated B cells, with IRF4 being itself a direct target of MYC transactivation.It is noteworthy that variations at IRF4 and 8q24-MYC are recognized risk factors for CLL 2,3 .Collectively, these findings are consistent with CLL GWAS SNPs mapping within regions of active chromatin state that exert effects on B-cell cis-regulatory networks. We investigated the genetic pathways between the gene products in proximity to the GWAS SNPs using the LENS pathway tool 25 .These gene products were primarily involved in immune response, BCR-mediated signalling, apoptosis and maintenance of chromosome integrity, as well as interconnectivity between the gene products (Fig. 5).Pathways that were enriched included those related to interferon signalling and apoptosis (Supplementary Data 3). Impact of risk SNPs on heritability of CLL.By fitting all SNPs from GWAS simultaneously using Genome-wide Complex Trait Analysis, the estimated heritability of CLL attributable to all common variation is 34% ( ± 5%), thus having potential to explain 57% of the overall familial risk.This estimate represents the additive variance and, therefore, does not include the potential impact of interactions or dominance effects or gene-environment interactions, having an impact on CLL risk.The currently identified risk SNPs (newly discovered and previously identified) only account for 25% of the additive heritable risk. ", "section_name": "Results", "section_num": null }, { "section_content": "Besides providing additional evidence for genetic susceptibility to CLL, the new and established risk loci identified further insights into the biological basis of CLL development.These loci annotate genes that participate in interconnecting cellular pathways, which are central to B-cell development.In particular, we note the involvement of BCR-mediated signalling with immune responses and apoptosis.Importantly, gene discovery initiatives can have an impact on the successful development of new therapeutic agents 26 .In this respect it is notable that Ibrutinib 27 (a BTK inhibitor) and Idelalisib 28 (a PI3KCD inhibitor) mediate their effects through interference of BCR signalling, and Venetoclax 29 targets the anti-apoptotic behaviour of BCL-2.The power of our GWAS to identify common alleles conferring relative risks of 1.2 or greater (such as the rs35923643 variant) is high (B80%).Hence, there are unlikely to be many additional SNPs with similar effects for alleles with frequencies greater than 0.2 in populations of European ancestry.In contrast, our analysis had limited power to detect alleles with smaller effects and/or MAFo0.1.Hence, further GWAS studies in concert with functional analyses should lead to additional insights into CLL biology and afford the prospect of development of novel therapies. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Ethics.Collection of patient samples and associated clinicopathological information was undertaken with written informed consent and relevant ethical review board approval at respective study centres in accordance with the tenets of the Declaration of Helsinki.Specifically, these centres are UK-CLL1 and UK-CLL2 Genome-wide association studies.The meta-analysis was based on six GWAS 2,6,7,9 (Supplementary Tables 1 and2).Briefly, the six GWAS comprised-UK-CLL1: 517 CLL cases and 2,698 controls; UK-CLL2: 1,403 CLL cases, 2,501 controls; Genetic Epidemiology of CLL (GEC) Consortium: 396 CLL cases and 296 controls; NHL GWAS Consortium: 1,851 CLL cases and 6,649 controls; UCSF: 214 CLL cases, 751 controls; Utah: 331 CLL cases, 420 controls. Quality control of GWAS.Standard quality-control measures were applied to the GWAS 31 .Specifically, individuals with low call rate (o95%) as well as all individuals evaluated to be of non-European ancestry (using the HapMap version 2 CEU, JPT/CHB and YRI populations as a reference) were excluded.For apparent first-degree relative pairs, we removed the control from a case-control pair; otherwise, we excluded the individual with the lower call rate.SNPs with a call rate o95% were excluded as were those with a MAF o0.01 or displaying significant deviation from Hardy-Weinberg equilibrium (that is, Po10 À 6 ).GWAS data were imputed to 410 million SNPs with the IMPUTE2 v2.3 software 32 using a merged reference panel consisting of data from 1000 Genomes Project (phase 1 integrated release 3 March 2012) 10 and UK10K (ref.11).Genotypes were aligned to the positive strand in both imputation and genotyping.Imputation was conducted separately for each study, and in each the data were pruned to a common set of SNPs between cases and controls before imputation.We set thresholds for imputation quality to retain potential risk variants with MAF40.005 for validation.Poorly imputed SNPs defined by an information measure o0.80 were excluded.Tests of association between imputed SNPs and CLL was performed using logistic regression under an additive genetic model in SNPTESTv2.5 (ref.33). The adequacy of the case-control matching and possibility of differential genotyping of cases and controls were formally evaluated using Q-Q plots of test statistics (Supplementary Fig. 1).The inflation factor l was based on the 90% leastsignificant SNPs 34 .Where appropriate, principal components, generated using common SNPs, were included in the analysis to limit the effects of cryptic population stratification that otherwise might cause inflation of test statistics. Eigenvectors for the GWAS data sets were inferred using smartpca (part of EIGENSOFT 35 ) by merging cases and controls with Phase II HapMap samples. Replication studies and technical validation.The 16 SNPs in the most promising loci were taken forward for de novo replication (Supplementary Table 2). The UK replication series comprised 645 cases collected through the NCLLC and Leicester Haematology Tissue Bank and 2,341 controls comprised 2,780 healthy individuals ascertained through the National Study of Colorectal Cancer (1999-2006; ref. 36).These controls were the spouses or unrelated friends of individuals with malignancies.None had a personal history of malignancy at the time of ascertainment.Both cases and controls were British residents and had selfreported European ancestry.The Mayo replication series comprised 407 newly diagnosed cases and 1,207 clinic-based controls from the Mayo Clinic CLL case-control study 37 .The eligibility criteria of the cases were age 20 years and older, consented within 9 months of their initial diagnosis at presentation to Mayo Clinic and no history of HIV.The eligibility criteria for the controls were age 20 years and older, a resident of Minnesota, Iowa or Wisconsin at the time of appointment at Mayo Clinic, no history of lymphoma or leukaemia and no history of HIV infection.Controls were frequency matched to the regional case distribution on 5-year age group, sex and geographic area.In silico replication was performed in 444 cases and 609 controls from International Cancer Genome Consortium (ICGC), and 226 cases and 228 controls from the WHI study 38,39 .The fidelity of imputation as assessed by the concordance between imputed and directly genotyped SNPs was examined in a subset of samples (Supplementary Table 5).Replication genotyping of UK samples was performed using competitive allele-specific PCR KASPar chemistry (LGC, Hertfordshire, UK); replication genotyping of Mayo samples was performed using Sequenom MassARRAY (Sequenom Inc., San Diego, CA, USA).Primers are listed in Supplementary Table 9.Call rates for SNP genotypes were 495% in each of the replication series.To ensure the quality of genotyping in all assays, at least two negative controls and duplicate samples (showing a concordance of 499%) were genotyped at each centre.To exclude technical artefacts in genotyping, we performed cross-platform validation of 96 samples and sequenced a set of 96 randomly selected samples from each case and control series to confirm genotyping accuracy.Assays were found to be performing robustly; concordance was 499%. Meta-analysis.Meta-analyses were performed using the fixed-effects inversevariance method based on the b estimates and s.e.'s from each study using META v1.6 (ref.40).Cochran's Q-statistic to test for heterogeneity and the I 2 statistic to quantify the proportion of the total variation due to heterogeneity were Each arm represents a functional annotation term, each arc represents an interaction between two proteins and the distance from the centre of the plot corresponds to a greater number of protein-protein interactions (higher degree of the node).The left arm represents proteins annotated as being involved in BCR signalling; the top arm represents proteins annotated as immune response; the right arm represents proteins involved in apoptosis; and the bottom arm represents proteins involved in DNA damage and chromosomal integrity.Selected proteins known to be involved in CLL risk are shown. calculated 41 .Using the meta-analysis summary statistics and LD correlations from a reference panel of the 1000 Genomes Project combined with UK10K we used Genome-wide Complex Trait Analysis to perform conditional association analysis 42 .Association statistics were calculated for all SNPs conditioning on the top SNP in each loci showing genome-wide significance.This is carried out in a step-wise manner. Analysis of exome-sequencing data.Previously published exome-sequencing data from 141 cases from 66 CLL families 43 were interrogated to search for deleterious (missense, nonsense, frameshift or splice site) variants within a genomic interval spanning all SNPs with LD r 2 40.2 with each index SNP.Positions resulting in protein-altering changes were identified using the Ensembl Variant Effect Predictor (version 78). Mutational status.IGVH mutation status was determined according to the BIOMED-2 protocols as described previously 44 .Sequence analysis was conducted using the Chromas software version 2.23 (Applied Biosystems) and the international immunogenetics information system database.In accordance with published criteria, we classified sequences with a germline identity of Z98% as unmutated and those with an identity of o98% as mutated. Association between genotype and patient outcome.To examine the relationship between SNP genotype and patient outcome, we analysed two patient series: (1) 356 patients from the UK Leukaemia Research Fund (LRF) CLL-4 trial 45 , which compared the efficacy of fludarabine, chlorambucil and the combination of fludarabine plus cyclophosphamide; (2) 377 newly diagnosed patients from Mayo Clinic who were prospectively followed.Cox-regression analysis was used to estimate genotype-specific hazard ratios and 95% CIs with overall survival.Statistical analyses were undertaken using R version 2.5.0. eQTL analysis.eQTL analyses were performed by examining the gene expression profiles of 452 CLL cases (Affymetrix Human Genome U219 Array) 46 .Additional data were obtained by querying publicly available eQTL mRNA expression data using MuTHER 47 , the Blood eQTL browser 48 and data from the GTEx consortium 49 .MuTHER contains expression data on LCLs, skin and adipose tissue from 856 healthy twins.The Blood eQTL browser contains expression data from 5,311 non-transformed peripheral blood samples.We used the whole-blood RNA-seq data from GTEx, which consisted of data from 338 individuals. Functional annotation.Novel risk SNPs and their proxies (that is, r 2 40.2 in the 1000 Genomes EUR reference panel) were annotated for putative functional effect based upon histone mark ChIP-seq/ChIPmentation data for H3K27ac, H3K4Me1 and H3K27Me3 from GM12878 (LCL) 18 and primary CLL cells 19 .We searched for overlap with 'super-enhancer' regions as defined by Hnisz et al. 21, restricting the analysis to the GM12878 cell line and CD19 þ B cells.We also interrogated ATAC-seq data from CLL cells 19 and primary B cells 20 .The novel risk SNPs and their proxies (r 2 40.2 as above) were intersected with regions of accessible chromatin in CLL cells, as defined by Rendeiro et al. 19 , which were used as a surrogate for likely sites of TF binding.SNPs falling within accessible sites (n ¼ 47) were taken forward to TF-binding motif analysis and were also annotated for genomic evolutionary rate profiling score 50 as well as bound TFs based on ENCODE project 18 ChIP-seq data. TF-binding disruption analysis.To determine whether the risk variants or their proxies were disrupting motif-binding sites, we used the motifbreakR package 22 .This tool predicts the effects of variants on TF-binding motifs, using position probability matrices to determine the likelihood of observing a particular nucleotide at a specific position within a TF-binding site.We tested the SNPs by estimating their effects on over 2,800 binding motifs as characterized by ENCODE 51 , FactorBook 52 , HOCOMOCO 53 and HOMER 54 .Scores were calculated using the relative entropy algorithm. TF and histone mark enrichment analysis.To examine enrichment in specific TF binding across risk loci, we adapted the variant set enrichment method of Cowper-Sal lari et al. 23 .Briefly, for each risk locus, a region of strong LD (defined as r 2 40.8 and D 0 40.8) was determined, and these SNPs were termed the associated variant set (AVS).TF ChIP-seq uniform peak data were obtained from ENCODE for the GM12878 cell line, which included data for 82 TF and 11 histone marks.For each of these marks, the overlap of the SNPs in the AVS and the binding sites was determined to produce a mapping tally.A null distribution was produced by randomly selecting SNPs with the same characteristics as the risk-associated SNPs, and the null mapping tally calculated.This process was repeated 10,000 times, and approximate P-values were calculated as the proportion of permutations where null mapping tally was greater or equal to the AVS mapping tally.An enrichment score was calculated by normalizing the tallies to the median of the null distribution.Thus, the enrichment score is the number of s.d.'s of the AVS mapping tally from the mean of the null distribution tallies. Heritability analysis.We used genome-wide complex trait analysis 42 to estimate the polygenic variance (that is, heritability) ascribable to all genotyped and imputed GWAS SNPs.SNPs were excluded based on low MAF (MAFo0.01),poor imputation (info score o0.4) and evidence of departure from Hardy Weinberg Equilibrium (HWE) (Po0.05).Individuals were excluded for poor imputation and where two individuals were closely related.A genetic relationship matrix of pairs of samples was used as input for the restricted maximum likelihood analysis to estimate the heritability explained by the selected set of SNPs.To transform the estimated heritability to the liability scale, we used the lifetime risk 55,56 for CLL, which is estimated to be 0.006 by SEER (http://seer.cancer.gov/statfacts/html/clyl.html).The variance of the risk distribution due to the identified risk loci was calculated as described by Pharoah et al. 57 , assuming that the relative risk when a first-degree relative has CLL is 8.5 (ref.1). Pathway analysis.To investigate the interaction between the gene products of the GWAS hits, we performed a pathway analysis.We selected the closest coding genes for the lead-associated SNPs and then performed pathway analysis using the LENS tool 25 , which identifies gene product and protein-protein interactions from HPRD 58 and BioGRID 59 .Enrichment of pathways was assessed using Fisher's exact test, comparing the overlap of the genes in the network with the genes in the pathway.Pathway data were obtained from REACTOME 60 .Cytoscape was used to perform network analyses 61 , and the Hive Plot was drawn using HiveR (academic.depauw.edu/Bhanson/HiveR/HiveR.html). ", "section_name": "Methods", "section_num": null } ]	[ { "section_content": "In the United Kingdom, Bloodwise provided funding for the study (LRF05001, LRF06002 and LRF13044) with additional support from Cancer Research UK (C1298/A8362 supported by the Bobby Moore Fund) and the Arbib Fund.G.P.S. is in receipt of a PhD studentship from The Institute of Cancer Research.The NCI/InterLymph NHL GWAS initiative was supported by the intramural programme of the Division of Cancer Epidemiology and Genetics, National Cancer Institute, US National Institutes of Health.ATBC-This research was supported in part by the Intramural Research Program of the NIH and the National Cancer Institute.In addition, this research was supported by U.S. Public Health Service contracts N01-CN-45165, N01-RC-45035, N01-RC-37004 and HHSN261201000006C from the National Cancer Institute, Department of Health and Human Services.BC-Canadian Institutes for Health Research (CIHR); Canadian Cancer Society; Michael Smith Foundation for Health Research.CPS-II-The Cancer Prevention Study-II (CPS-II) Nutrition Cohort is supported by the American Cancer Society.Genotyping for all CPS-II samples was supported by the Intramural Research Program of the National Institutes of Health, NCI, Division of Cancer Epidemiology and Genetics.We also acknowledge the contribution to this study from central cancer registries supported through the Centers for Disease Control and Prevention National Program of Cancer Registries, and cancer registries supported by the National Cancer Institute Surveillance Epidemiology and End Results program.ELCCS-Leukemia and Lymphoma Research.ENGELA-Association pour la Recherche contre le Cancer (ARC), Institut National du Cancer (INCa), Fondation de France, Fondation contre la Leuce ´mie, Agence nationale de se ´curite ´sanitaire de l'alimentation, de l'environnement et du travail (ANSES).EPIC-Coordinated Action (Contract #006438, SP23-CT-2005-006438); HuGeF (Human Genetics Foundation), Torino, Italy; Cancer Research UK.EpiLymph-European Commission (grant references QLK4-CT-2000-00422 and FOOD-CT-2006-023103); the Spanish Ministry of Health (grant references CIBERESP, PI11/01810, PI14/01219, RCESP C03/09, RTICESP C03/10 and RTIC RD06/0020/0095), the Marato de TV3 Foundation (grant reference 051210), the Age `ncia de Gestio ´d'AjutsUniversitarisi de Recerca-Generalitat de Catalunya (grant reference 2014SRG756), who had no role in the data collection, analysis or interpretation of the results; the NIH (contract NO1-CO-12400); the Compagnia di San Paolo-Programma Oncologia; the Federal Office for Radiation Protection grants StSch4261 and StSch4420, the Jose ´Carreras Leukemia Foundation grant DJCLS-R12/23, the German Federal Ministry for Education and Research (BMBF-01-EO-1303); the Health Research Board, Ireland, and Cancer Research Ireland; Czech Republic supported by MH CZ-DRO (MMCI, 00209805) and RECAMO, CZ.1.05/2.1.00/03.0101;Fondation de France and Association de Recherche Contre le Cancer.GEC/Mayo GWAS-National Institutes of Health (CA118444, CA148690, CA92153).Intramural Research Program of the NIH, National Cancer Institute.Veterans Affairs Research Service.Data collection for Duke University was supported by a Leukemia and Lymphoma Society Career Development Award, the Bernstein Family Fund for Leukemia and Lymphoma Research and the National Institutes of Health (K08CA134919), National Center for Advancing Translational Science (UL1 TR000135).HPFS-The HPFS was supported in part by National Institutes of Health grants CA167552, CA149445 and CA098122.We would like to thank the participants and staff of the Health Professionals Follow-up Study for their valuable contributions as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY.We assume full responsibility for analyses and interpretation of these data.Iowa-Mayo SPORE-NCI Specialized Programs of Research Excellence (SPORE) in Human Cancer (P50 CA97274); National Cancer Institute (P30 CA086862, P30 CA15083); Henry J. Predolin Foundation.Italian GxE-Italian Association for Cancer Research (AIRC, Investigator Grant 11855; PC); Fondazione Banco di Sardegna 2010-2012 and Regione Autonoma della Sardegna (LR7 CRP-59812/2012; MGE).Mayo Clinic Case-Control-National Institutes of Health (R01 CA92153); National Cancer Institute (P30 CA015083).MCCS-The Melbourne Collaborative Cohort Study recruitment was funded by VicHealth and Cancer Council Victoria.The MCCS was further supported by Australian NHMRC grants 209057, 251553 and 504711 and by infrastructure provided by Cancer Council Victoria.Cases and their vital status were ascertained through the Victorian Cancer Registry (VCR).MD Anderson-Institutional support to the Center for Translational and Public Health Genomics.MSKCC-Geoffrey Beene Cancer Research Grant, Lymphoma Foundation (LF5541); Barbara K. Lipman Lymphoma Research Fund (74419); Robert and Kate Niehaus Clinical Cancer Genetics Research Initiative (57470); U01 HG007033; ENCODE; U01 HG007033.R21 CA178800.NCI-SEER-Intramural Research Program of the National Cancer Institute, National Institutes of Health and Public Health Service (N01-PC-65064, N01-PC-67008, N01-PC-67009, N01-PC-67010, N02-PC-71105).NHS-The NHS was supported in part by National Institutes of Health grants CA186107, CA87969, CA49449, CA149445 and CA098122.We would like to thank the participants and staff of the Nurses' Health Study for their valuable contributions as well as the following state cancer registries for their help: A.L., A.Z., A.R., C.A., C.O., C.T., D.E., F.L., G.A., I.D., I.L., I.N., I.A., K.Y., L.A., M.E., M.D., M.A., M.I., N.E., N.H., N.J., N.Y., N.C., N.D., O.H., O.K., O.R., P.A., R.I., S.C., T.N., T.X., V.A., W.A. and W.Y. The authors assume full responsibility for analyses and interpretation of these data.NSW-NSW was supported by grants from the Australian National Health and Medical Research Council (ID990920), the Cancer Council NSW and the University of Sydney Faculty of Medicine.NYU-WHS-National Cancer Institute (R01 CA098661, P30 CA016087); National Institute of Environmental Health Sciences (ES000260).PLCO-This research was supported by the Intramural Research Program of the National Cancer Institute and by contracts from the Division of Cancer Prevention, National Cancer Institute, NIH, DHHS.SCALE-Swedish Cancer Society (2009/659).Stockholm County Council (20110209) and the Strategic Research Program in Epidemiology at Karolinska Institute.Swedish Cancer Society grant (02 6661).National Institutes of Health (5R01 CA69669-02); Plan Denmark.UCSF2-The UCSF studies were supported by the NCI, National Institutes of Health, CA1046282, CA154643, CA45614, CA89745, CA87014.The collection of cancer incidence data used in this study was supported by the California Department of Health Services as part of the statewide cancer reporting programme mandated by California Health and Safety Code Section 103885; the National Cancer Institute's Surveillance, Epidemiology and End Results Program under contract HHSN261201000140C awarded to the Cancer Prevention Institute of California, contract HHSN261201000035C awarded to the University of Southern California, and contract HHSN261201000034C awarded to the Public Health Institute; and the Centers for Disease Control and Prevention's National Program of Cancer Registries, under agreement #1U58 DP000807-01 awarded to the Public Health Institute.The ideas and opinions expressed herein are those of the authors, and endorsement by the State of California, the California Department of Health Services, the National Cancer Institute or the Centers for Disease Control and Prevention or their contractors and subcontractors is not intended nor should be inferred.UTAH-National Institutes of Health CA134674.Partial support for data collection at the Utah site was made possible by the Utah Population Database (UPDB) and the Utah Cancer Registry (UCR).Partial support for all data sets within the UPDB is provided by the Huntsman Cancer Institute (HCI) and the HCI Comprehensive Cancer Center Support grant, P30 CA42014.The UCR is supported in part by NIH contract HHSN261201000026C from the National Cancer Institute SEER Program with additional support from the Utah State Department of Health and the University of Utah.WHI-WHI investigators are: Program Office (National Heart, Lung, and Blood Institute, Bethesda, Maryland)-Jacques Rossouw, Shari Ludlam, Dale Burwen, Joan McGowan, Leslie Ford and Nancy Geller; Clinical Coordinating Center (Fred Hutchinson Cancer Research Center, Seattle, WA)-Garnet Anderson, Ross Prentice, Andrea LaCroix and Charles Kooperberg; Investigators and Academic Centers (Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA)-JoAnn E. Manson; (MedStar Health Research Institute/ Howard University, Washington, DC, USA) Barbara V. Howard; (Stanford Prevention Research Center, Stanford, CA, USA) Marcia L. Stefanick (The Ohio State University, Columbus, OH, USA); Rebecca Jackson (University of Arizona, Tucson/Phoenix, AZ, USA); Cynthia A. Thomson; (University at Buffalo, Buffalo, NY, USA); Jean Wactawski-Wende (University of Florida, Gainesville/Jacksonville, FL, USA); Marian Limacher (University of Iowa, Iowa City/Davenport, IA, USA); Robert Wallace (University of Pittsburgh, Pittsburgh, PA, USA); Lewis Kuller (Wake Forest University School of Medicine, Winston-Salem, NC, USA); Sally Shumaker WHI Memory Study (Wake Forest University School of Medicine, Winston-Salem, NC, USA) Sally Shumaker. The WHI programme is funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, U.S. Department of Health and Human Services through contracts HHSN268201100046C, HHSN268201100001C, HHSN268201100002C, HHSN268201100003C, HHSN268201100004C and HHSN271201100004C.YALE-National Cancer Institute (CA62006); National Cancer Institute (CA165923).The Spanish replication study was supported by the Spanish Ministry of Economy and Competitiveness through the Instituto de Salud Carlos III (FIS PI13/01136; International Cancer Genome Consortium-Chronic Lymphocytic Leukemia Genome Project).We thank L. Padyukov (Karolinska Institutet) and the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) group for providing control samples from the Swedish population for the Swedish replication study.MCCS cohort recruitment was funded by VicHealth and Cancer Council Victoria.The MCCS was further supported by Australian NHMRC grants 209057, 251553 and 504711, and by infrastructure provided by Cancer Council Victoria.Cases and their vital status were ascertained through the Victorian Cancer Registry (VCR) and the Australian Institute of Health and Welfare (AIHW), including the National Death Index and the Australian Cancer Database.This study makes use of data generated by the Wellcome Trust Case Control Consortium.A full list of the investigators who contributed to the generation of the data is available in www.wtccc.org.uk.Funding for the project was provided by the Wellcome Trust under award 076113.We are grateful to all investigators and all the patients and individuals for their participation.We also thank the clinicians, other hospital staff and study staff that contributed to the blood sample and data collection for this study. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Data availability.Genotype data that support the findings of this study have been deposited in the database of Genotypes and Phenotypes (dbGAP) under accession code phs000802.v2.p1 and in the European Genome-phenome Archive (EGA) under accession codesEGAS00001000090, EGAD00001000195, EGAS00001000108, EGAD00000000022 and EGAD00000000024. Transcriptional profiling data from the MuTHER consortium that support the findings of this work have been deposited in the European Bioinformatics Institute (Part of the European Molecular Biology Laboratory, EMBL-EBI) under accession code E-TABM-1140.Data from Blood eQTL have been deposited in the EBI-EMBL under accession codes E-TABM-1036, E-MTAB-945 and E-MTAB-1708.GTEx data are deposited in dbGaP under accession code phs000424.v6.p1.The remaining data are contained within the paper and its Supplementary files or are available from the authors upon reasonable request. ", "section_name": "", "section_num": "" }, { "section_content": "R.H. and S.L.S. developed the project and provided overall project management; R.H., S.L.S., P.J.L., H.E.S. and G.P.S. drafted the manuscript.At the ICR: P.J.L., G.P.S. and H.E.S. performed bioinformatic and statistical analyses; H.E.S. performed project management and supervised genotyping; G.P.S. and A.H. performed sequencing and genotyping.In Newcastle, J.M.A. and D.J.A. conceived of the NCLLC; J.M.A. obtained financial support, supervised laboratory management and oversaw genotyping of cases with NCLLC; N.J.S. and H.M. performed sample management of cases; A. Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications Competing financial interests: The authors declare no competing financial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/ ", "section_name": "Author contributions", "section_num": null }, { "section_content": "R.H. and S.L.S. developed the project and provided overall project management; R.H., S.L.S., P.J.L., H.E.S. and G.P.S. drafted the manuscript.At the ICR: P.J.L., G.P.S. and H.E.S. performed bioinformatic and statistical analyses; H.E.S. performed project management and supervised genotyping; G.P.S. and A.H. performed sequencing and genotyping.In Newcastle, J.M.A. and D.J.A. conceived of the NCLLC; J.M.A. obtained financial support, supervised laboratory management and oversaw genotyping of cases with NCLLC; N.J.S. and H.M. performed sample management of cases; A. ", "section_name": "Author contributions", "section_num": null }, { "section_content": "Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications Competing financial interests: The authors declare no competing financial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/ ", "section_name": "Additional information", "section_num": null } ]
10.1186/s12885-019-5681-6	Probabilistic modeling of personalized drug combinations from integrated chemical screen and molecular data in sarcoma	<jats:title>ABSTRACT</jats:title><jats:p>Cancer patients with advanced disease exhaust available clinical regimens and lack actionable genomic medicine results, leaving a large patient population without effective treatments options when their disease inevitably progresses. To address the unmet clinical need for evidence-based therapy assignment when standard clinical approaches have failed, we have developed a probabilistic computational modeling approach which integrates sequencing data with functional assay data to develop patient-specific combination cancer treatments. This computational modeling approach addresses three major challenges in personalized cancer therapy, which we validate across multiple species via computationally-designed personalized synergistic drug combination predictions, identification of unifying therapeutic targets to overcome intra-tumor heterogeneity, and mitigation of cancer cell resistance and rewiring mechanisms. These proof-of-concept studies support the use of an integrative functional approach to personalized combination therapy prediction for the population of high-risk cancer patients lacking viable clinical options and without actionable DNA sequencing-based therapy.</jats:p>	[ { "section_content": "Despite decades of advancements in cancer treatment, over 600,000 patients with solid tumors die annually in North America [1], including approximately 5000 sarcoma-related deaths.The population of high-risk, late-stage, recurrent, rare or refractory cancer patients who have exhausted standard clinical pathways and lack further treatment options represents a major unmet clinical need.Currently, DNA sequencing of tumors for druggable mutations leaves approximately 60% of patients without an actionable result [2,3].Additionally, in many cases, single drug therapy fails to provide sustainable disease control [4].A critical missing element in personalized cancer therapy design is the lack of effective methodologies for model-based prediction, design, and prioritization of patient-specific drug combinations, especially in the presence of limited tumor tissue material. Numerous approaches to computational modeling of drug sensitivity and therapy assignment exist, in part to address ambiguity in DNA sequencing results [2,5].These approaches are primarily based on gene expression [6], or a combination of genomic and epigenomic data [7].For instance, 1) integrative genomic models using Elastic Net regression techniques have been developed from large datasets such as the Cancer Cell Line Encyclopedia (CCLE) [8] database; 2) integrative models using Random Forests with Stacking [9,10] to integrate multiple genetic data sets for sensitivity prediction; and 3) a team science based sensitivity prediction challenge produced independent models integrating multiple data types for sensitivity prediction [11]; despite 44 individual models and a \"wisdom of crowds\" approach merging the top-ranked predictive models together, none of the approaches surpassed 70% predictive accuracy [11] falling short of a reasonable accuracy threshold for clinical utility.Some recent work has focused on the use of functional data for therapy selection, such as 1) the use of microfluidics to test multiple drugs efficiently on primary patient samples [12], 2) the use of shRNA libraries to predict drug combinations for heterogenous tumor populations [13], and 3) a re-analysis of the CCLE database used machine learning models integrating functional response data to improve sensitivity prediction accuracy over molecular data-based Elastic Net models [14].Integration of functional data may improve overall predictive accuracy over solely molecular data-based predictive models, especially for individual patient samples, emphasizing the need for improved drug sensitivity prediction to enable patient-specific therapy design. To address the need for accurate prediction of drug sensitivity and design of multi-drug combinations, we previously developed a functional drug sensitivity-based modeling approach termed Probabilistic Target Inhibition Maps (PTIMs) [14][15][16][17].The base PTIM methodology integrates quantified drug-target inhibition information (EC 50 values) and log-scaled experimental drug sensitivities (IC 50 values) to identify mechanistic target combinations explaining drug sensitivity data.PTIM modeling improved predictive accuracy over Elastic Net models from the CCLE dataset [14], and has guided in silico validation experiments from primary canine osteosarcoma cell models [14,[16][17][18] and in vitro validation experiments [19] on diffuse intrinsic pontine glioma (DIPG) cell models.Herein, we present proof-ofconcept validation experiments of the integrative PTIM pipeline (Fig. 1) using soft tissue sarcoma as a paradigm.Each validation experiment applies PTIM combination therapy design to address one of three critical unmet needs in cancer treatment: 1) selection of functional evidence-based synergistic drug combinations, validated in murine alveolar rhabdomyosarcoma (aRMS); 2) consensus modeling of multi-site drug sensitivity data to overcome intra-tumor heterogeneity, validated in epithelioid sarcoma (EPS); and 3) resistance abrogation by targeting of parallel biological pathways, validated in undifferentiated pleomorphic sarcoma (UPS). ", "section_name": "Background", "section_num": null }, { "section_content": "", "section_name": "Methods", "section_num": null }, { "section_content": "The mouse primary tumor cell culture U23674 was established from a tumor at its site of origin in a genetically engineered Myf6Cre,Pax3:Foxo1,p53 mouse bearing alveolar rhabdomyosarcoma (aRMS) as previously described [20].In brief, the tumor was minced and digested with collagenase (10 mg/ml) overnight at 4 °C.Dissociated cells were then incubated in Dulbecco's Modified Eagle's Medium (DMEM) (11995-073; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (26,140,079; Thermo Fisher Scientific) and 1% penicillin-streptomycin (15140-122; Thermo Fisher Scientific) in 5% CO 2 at 37 °C. The human epithelioid sarcoma (EPS) sample PCB490 was collected from a patient undergoing planned surgical resection.Tumor tissue was partitioned into 5 distinct regions, minced and digested with collagenase type IV (10 mg/ml) overnight at 4 °C.The dissociated cells were then incubated in RPMI-1640 (11875-093; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in 5% CO 2 at 37 °C.Sections 3, 4, and 5 (PCB490-3, PCB490-4, PCB490-5) successfully grew in culture.Samples from each region were also sent to The Jackson Laboratory (JAX) for patient-derived xenograft (PDX) model establishment.Cultures were maintained at low passage to minimize biological variation from the original patient tumor.Remaining Fig. 1 Schematic representation of experimental and computational approach to personalized combination targeted therapy predictions.Following tumor extraction and culture establishment, biological data is generated (e.g., chemical screening, transcriptome sequencing, exome sequencing, siRNA interference screening and phosphoproteomic analysis) and used as input for PTIM modeling.To briefly explain the graphical model representation, targets A and B denote two independent single points of failure.Targets C and D denote parallel targets, which independently are not predicted to be effective, but together will be synergistic and lead to significant cell growth inhibition.Targets A, B, and the C-D parallel block are in series and may target independent pathways.Series blocks, when inhibited together, may abrogate cancer resistance mechanisms by knockdown of independent pathways.Model sensitivity scores for gene target combinations are used to design and rank follow-up in vitro validation and in vivo validation experiments.The \"Exome-Seq\" representative images was adapted from an image on the Wikipedia Exome sequencing article originally created by user SarahKusala and available under Creative Commons 3.0 license.An unaltered portion of the image was used.The mouse image used is public domain and accessed through Bing image search at the following weblink: http://img.res.meizu.com/img/download/uc/27/83/20/60/00/2783206/w100h100tumor pieces were snap frozen for future DNA, RNA and protein isolation. The human EPS sample PCB495 was received through the CCuRe-FAST tumor bank program.To create the cell cultures from the PCB495 primary tumor, the tumor was minced and digested with collagenase (10 mg/ml) overnight at 4 °C.The dissociated cells were then incubated in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in 5% CO 2 at 37 °C. The human undifferentiated pleomorphic sarcoma (UPS) PCB197 was received through the CCuRe-FAST tumor bank program.To create the cell cultures from the PCB197 primary tumor, the tumor was minced and digested with collagenase (10 mg/ml) overnight at 4 °C.The dissociated cells were then incubated in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in 5% CO 2 at 37 °C. All human tissue samples were acquired through the Childhood Cancer Registry for Familial and Sporadic Tumors (CCuRe-FAST) tumor banking program.All patients enrolled in CCuRe-FAST provided informed consent.All aspects of the study were reviewed and approved by the Oregon Health & Science University (OHSU) Institutional Review Board (IRB).Patient data and clinical and pathologic information are maintained in a de-identified database. The canine UPS sample S1-12 was obtained from Oregon State University's (OSU) College of Veterinary Medicine.OSU Institutional Animal Care and Use Committee (IACUC) approval was obtained for procurement of the tissue.To establish S1-12 cell culture, tumor tissue was minced and digested with collagenase (10 mg/ ml) overnight at 4 °C.The dissociated cells were then incubated in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in 5% CO 2 at 37 °C. ", "section_name": "Cell model establishment", "section_num": null }, { "section_content": "Tumor tissue and cells from PCB490-1,2, and 5 were lysed in radioimmunoprecipitation (RIPA) buffer containing both protease and phosphatase inhibitors (Sigma Aldrich, St. Louis, MO).Lysates were homogenized and clarified by centrifugation at 14,000 rpm for 10 min.Thirty μg of protein was electrophoresed in 7.5% polyacrylamide gels, transferred to PVDF membranes for immunoblot analysis with mouse anti-BAF47 antibody (cat.612,110, BD Biosciences, San Jose, CA) and mouse anti-β-actin antibody (cat.A1978, Sigma Aldrich), and developed by chemiluminescence (cat.170-5061, BioRad Clarity Western ECL Substrate, Hercules, CA) per the manufacturer's protocol. ", "section_name": "Immunoblotting of PCB490", "section_num": null }, { "section_content": "The VA-ES-BJ (Accession CVCL_1785) cell line was purchased commercially (cat# CRL-2138, ATCC, Manassas, VA).The cell line VA-ES-BJ has been validated before shipment by STR profile and mycoplasma testing.The cell line was used for the experiments directly after reception of the cell line. The ESX cell line was provided by author TT [21].The FU-EPS-1 (Accession CVCL_E311) cell line was provided by author JNishio [22]. Neither ESX nor FU-EPS-1 have available STR validation profiles, and so comparison to a standard STR profile cannot be performed.However, both cell lines were checked for INI1 loss consistent with EPS cell lines.Cell lines were tested for mycoplasma with the Lonza MycoAlert Plus test kit.Cat.LT07-703, Lonza Bioscience, Singapore). 3 , the tumor was harvested and divided into 3-5 mm 3 fragments.Fragments were implanted into five 6-8-week-old female NSG mice for expansion to P1.Other fragments were sent for quality control assessment (see below).The remaining fragments were cryopreserved in 10% DMSO.When P1 tumors reached 1000mm 3 they were harvested and divided into quarters: ¼ for quality control, ¼ snap frozen for genomics, ¼ placed into RNALater (Ambion) for RNA-seq, and the remaining ¼ divided into 3-5 mm 3 pieces and cryopreserved in 10% DMSO. ", "section_name": "Cell lines", "section_num": null }, { "section_content": "The quality control procedures employed for PDX model development included testing the patient tumor for LCMV (lymphocytic choriomeningitis virus), bacterial contamination, and tumor cell content.The engrafted tumors at P0 and P1 were DNA fingerprinted using a Short Tandem Repeat (STR) assay to ensure model provenance in subsequent passages. Model details available online at: http://tumor.informatics.jax.org/mtbwi/pdxDetails.do?modelID=J000078604 Immunohistochemistry (IHC) for human CD45 (IR75161-2, Agilent Technologies) was performed on paraffin embedded blocks of engrafted tumors to identify cases of lymphomagenesis which have been reported previously in PDXs.IHC for human ki67 (IR62661-2, Agilent Technologies) was used to ensure the propagated tumors were human in origin.H&E sections of engrafted tumors were reviewed by a board-certified pathologist (RGE) to evaluate concordance of the morphological features of the engrafted tumor to the patient tumor.Further, tissue was stained with vimentin (IR63061-2, Agilent Technologies) to confirm human origin. Model information is publicly accessible at: http:// tumor.informatics.jax.org/mtbwi/pdxSearch.do ", "section_name": "Patient derived xenograft (PDX) model development", "section_num": null }, { "section_content": "Four chemical screens were used to generate functional drug screening data.The first screen was a custom 60 agent chemical screen of well-characterized target inhibitors denoted the Pediatric Preclinical Testing Initiative Screen Version 2.1 (PPTI screen).Chemical concentrations of agents in all chemical screens were either [10 nM, 100 nM, 1 μM, 10 μM] or [100 nM, 1 μM, 10 μM, 100 μM] depending on compound activity range.Fifty-four of the 60 drugs on the chemical screen have a published quantified drug-target inhibition profile. The second screen was a custom 60 agent chemical screen denoted Drug Screen V3 consisting of a variety of small molecule kinase inhibitors, epigenetic target inhibitors, and cell cycle inhibitors.Fifty-two of 60 drugs on the chemical screen have a published drug-target inhibition profile. The third chemical screen was a GlaxoSmithKline open access Orphan Kinome-focused chemical screen (denoted GSK screen) consisting of 402 novel and newly characterized tool compounds [23] with target inhibition profiles quantified by Nanosyn Screening and Profiling Services.Drug-target interaction was assayed over 300 protein targets for each of the 402 compounds.The compounds were tested at 100 nM and 10 μM concentrations to bracket the drug-target EC 50 values.The final EC 50 values used for analysis of the chemical screen results were inferred from the available data using hill curve fitting to predict the 50% inhibition point. The final screen was a Roche-developed open access chemical screen (denoted Roche screen) consisting of 223 novel kinase inhibitor compounds [24].Roche screen compounds had a mixture of quantified or qualified drug-target inhibition profiles, though drug-target inhibition profiles were made available only for sensitive compounds. Cell cultures were plated in 384-well plates at a seeding density of 5000 cells per well onto gradated concentrations of drug screen compounds.Cells were incubated in model-specific culture media at 37 °C, with 5% CO 2 , for 72 h.Cell viability was assessed by CellTiter-Glo® Luminescent Cell Viability Assay (cat.G7570, Promega, Madison, WI) per manufacturer's protocol.Luminescence was measured using a BioTek Synergy HT plate reader (BioTek, Winooski, VT).Single agent IC 50 values were determined using a hill curve-fitting algorithm with variable hill slope coefficients performed in Microsoft Excel.Manual curation and re-fitting of the results was performed before results were finalized.U23674 primary tumor culture was assayed via three drug screens: PPTI drug screen, GSK drug screen, and the Roche drug screen (Additional files 1, 2, 3: Figures S1-S3 and Additional files 15, 16, 17, 18, 19, 20, 21: Tables S1-S7).S1-12 primary tumor culture was screened using the PPTI screen (Additional file 36: Table S22).PCB197 primary tumor culture was screened using the PPTI screen (Additional file 36: Table S22).PCB490-3, PCB490-4, PCB490-5 primary cultures were screened with Drug Screen V3 and the Roche drug screen (Fig. 3, Additional files 30, 31: Tables S15 andS16).Cell lines ESX, FU-EPS-1, and VA-ES-BJ were screened with Drug Screen V3 (Additional file 35: Table S21).PCB495 primary culture was screened with Drug Screen V3 (Additional file 35: Table S21). ", "section_name": "Chemical screens", "section_num": null }, { "section_content": "U23674 drug combination validation experiments were guided by GlaxoSmithKline chemical screen PTIM models.Single agent validations to calculate independent drug efficacy were performed at dosages in the range of 5 nM to 100 μM to bracket IC 50 and IC 25 dosage values; for combination experiments, the IC 25 dosage for one agent was tested in combination with gradated dosages (5 nM to 100 μM) of the complementary agent, and vice versa.Single agent and combination agent validation experiments were performed at passage 5. CI values were generated using the CompuSyn software tool.Effect values for CompuSyn monotherapy and combination therapy were determined by mean cell death based on n = 3 technical replicates with n = 4 replicates for the following treatment conditions: OSI-906, GDC-0941, OSI-906 + GDC-0941 (OSI-906 at IC 25 + GDC-0941 at varying dosage, OSI-906 at varying dosage + GDC-0941 at IC 25 ).CompuSyn CI values were calculated using the non-constant combination setting [25] (Additional file 28: Table S14). We performed low-dose validation experiments to verify PTIM-identified synergistic mechanisms of action; reduced dosages of the combination agents were set to 5 times the EC 50 value for the predicted target (175 nM OSI-906, 50 nM GDC-0941).CompuSyn CI values to validate the mechanism of synergy were calculated using the non-constant combination setting [25] (Additional file 28: Table S14). In both regular dose and low dose experiments, CI values are reported only for functionally relevant dosages, i.e. dosages between the drug target's EC 50 and the drug's maximum achievable human clinical dosage (C max ).For OSI-906, the functional range is approximately [10 nM, 5 μM] (mouse pharmacokinetics: ~16 μM C max , 6.16 μM C ss ; human pharmacokinetics: 1.481 μM C max , 720 nM C ss ).For GDC-0941, the functional range is approximately [5 nM, 1 μM] (mouse pharmacokinetics: ~12 μM C max , 1.59 μM C ss , human pharmacokinetics: ~1.481 μM C max , 720 nM C ss ).CI values outside these ranges are denoted as N/A in Additional file 28: Table S14. ", "section_name": "U23674 drug combination studies and calculation of combination index (CI)", "section_num": null }, { "section_content": "Somatic point mutations were identified using the Genome Analysis Toolkit [26] (GATK, version 3.5.0)from the Broad Institute.Captured DNA libraries were sequenced with the Illumina HiSeq 1000 in paired-end mode.The reads that passed the Illumina BaseCall chastity filter were used for subsequent analysis.The mate pairs were pooled and mapped as single reads to the NCBI GRCm38/mm10 reference genome using the Burrows-Wheeler Aligner [27] (version 0.7.12), with shorter split hits marked as secondary to ensure compatibility with downstream tools.Identified PCR duplicates, defined as reads likely originating from the same original DNA fragments, were removed using Picard Tools MarkDuplicates (version 1.133).Mapping artifacts introduced during initial mapping are realigned using the GATK IndelRealigner, and base quality score recalibration to empirically adjust quality scores for variant calling was performed by the GATK BaseRecalibrator tool.The same process was used to process both the tumor sample and the matched normal tail sample.Variant discovery was performed by MuTect2 [28], with the NCBI GRCm38/mm10 dbSNP database used to filter known polymorphisms present in the paired sample.Variant annotation and effect prediction was performed using SnpEff [29] using the GRCm38.81database.Only medium and high impact effect variants are considered for the purpose of downstream analysis and reporting in figures.Exome analysis protocol is based on the GATK Best Practices protocol. VarScan2 was used for copy number variation analysis of the paired tumor-normal data [30].The Burrows-Wheeler Aligner was used to align the tumor and normal samples to NCBI GRCm38/mm10 reference genome as described previously.Samtools (version 0.1.19)mpileup tool with minimum mapping quality of 10 was used to generate the pileup file required by the VarScan2 copycaller function; log 2 exon coverage ratio data from copycaller was segmented using DNAcopy with the undo.splits= \"sdundo\" parameter, and deviation from the null hypothesis set above 3 standard deviations.Genes in segments with segment mean above 0.25 or below -0.25 and with p-value below 1e-10 were called as gained or lost, respectively.Copy number variation analysis protocol was partly based on the VarScan2 user manual [31]. ", "section_name": "U23674 exome sequencing analysis", "section_num": null }, { "section_content": "RNA sequencing was performed on a low-passage U23674 culture, and on the control sample consisting of regenerating mouse muscle tissue following cardiotoxin injury in vivo.The paired-end raw reads were aligned to the NCBI GRCm38/mm10 reference mouse genome using TopHat version 2.0.9 [32] using Bowtie2 as the short-read aligner.Up to two alignment mismatches were permitted before a read alignment was discarded.The aligned reads were assembled into transcripts using Cufflinks version 2.1.1 [33].Differential gene expression of tumor sample vs. control was performed by Cuffdiff using standard parameters.RNA analysis protocol was largely based on the approach described in the Tophat2 publication [34].Quantified gene expression is provided in Additional file 23: Table S9. ", "section_name": "U23674 RNA deep sequencing analysis", "section_num": null }, { "section_content": "Somatic point mutations were identified using the Genome Analysis Toolkit [26] (GATK, version 3.8.0)from the Broad Institute.Captured DNA libraries were sequenced in paired-end mode via the BGISeq 500 system at Beijing Genomics Institute.The reads that passed the Illumina BaseCall chastity filter were used for subsequent analysis.The mate pairs were pooled and mapped as single reads to the NCBI GRCh38 reference genome using the Burrows-Wheeler Aligner [27] (version 0.7.12), with shorter split hits marked as secondary to ensure compatibility with downstream tools.Identified PCR duplicates, defined as reads likely originating from the same original DNA fragments, were removed using Picard Tools MarkDuplicates (version 1.133).Mapping artifacts introduced during initial mapping are realigned using the GATK IndelRealigner, and base quality score recalibration to empirically adjust quality scores for variant calling was performed by the GATK BaseRecalibrator tool.The same process was used to process both the tumor sample and the matched normal sample.Variant discovery was performed by MuTect2 [28], with the NCBI GRCh38 dbSNP database used to filter known polymorphisms present in the paired sample.Variant annotation and effect prediction was performed using SnpEff [29] using the GRCh38.87database.Only medium and high impact variants are considered for the purpose of downstream analysis and reporting in figures.Exome analysis protocol is based on the GATK Best Practices protocol. VarScan2 was used for copy number variation analysis of the paired tumor-normal data [30].The Burrows-Wheeler Aligner was used to align the tumor and normal samples to NCBI GRCh38 reference genome as described previously.Samtools (version 1.6) mpileup tool with minimum mapping quality of 10 was used to generate the pileup file required by the VarScan2 copycaller function; log 2 exon coverage ratio data from copycaller was segmented using DNAcopy with the undo.splits= \"sdundo\" parameter, and deviation from the null hypothesis set above 3 standard deviations.Genes in segments with segment mean 2 standard deviations above or below ±0.5 and with p-value below 1e-10 were called as gained or lost, respectively.Copy number variation analysis protocol was partly based on the VarScan2 user manual [31]. ", "section_name": "PCB490 exome sequencing analysis", "section_num": null }, { "section_content": "The PCB490 transcriptome library was sequenced with the Illumina HiSeq 2500 in paired-end mode.The reads that passed the chastity filter of Illumina BaseCall software were used for subsequent analysis.The paired-end raw reads for each RNA-seq sample were aligned to the UCSC hg38 reference human genome using Bowtie2 as the short-read aligner [32] using, allowing up two alignment mismatches before a read alignment was discarded.The aligned reads were assembled into transcripts using Cufflinks version 2.1.1 [33] and quantification was performed with Cuffquant [33].RNA analysis protocol was adapted from the approach described in the original TopHat2 publication [34] (Additional file 33: Table S19). ", "section_name": "PCB490 RNA deep sequencing analysis", "section_num": null }, { "section_content": "U23674 underwent functional single gene knockdown (siRNA interference screen, Additional file 24: Table S10), however siRNA results were inconsistent with drug screening data (Additional file 25: Table S11) and are thus relegated to the supplement. To assess the contribution of individual receptor tyrosine kinases to survival of U23674, we performed RAPID siRNA knockdown screening of U23674.Efficacy of single target knockdown of 85 members of the mouse tyrosine kinase family was performed as previously described [35].Target sensitivity was determined by resulting cell viability quantified using an MTT assay (M6494; Thermo Fisher Scientific, Waltham, MA, USA).Targets with viability two standard deviations below the mean were identified as high-importance targets [35] (Additional file 24: Table S10). ", "section_name": "RAPID siRNA screen of U23674", "section_num": null }, { "section_content": "U23674 underwent phosphoproteome quantification (Kinexus phosphoproteomics analysis, Additional file 26: Table S12), however phosphoproteomics results were inconsistent among sample replicates and are thus relegated to the supplement. To identify differentially phosphorylated protein targets, phosphoproteomics assays (Kinexus, Vancouver, British Columbia, Canada) were used to compare two duplicate cell lysates from U23674 against two duplicate cell lysates from regenerating muscle tissue acting as normal control.To perform the phosphoproteomics analyses, 50 μg of protein lysate from each sample was covalently labeled with a proprietary fluorescent dye.Free dye molecules were removed by gel filtration.After blocking non-specific binding sites on the array, an incubation chamber was mounted onto the microarray to permit the loading of related samples side by side on the same chip.Following sample incubation, unbound proteins were washed away.Each array produces a pair of 16-bit images, which are captured with a Perkin-Elmer ScanArray Reader laser array scanner.Signal quantification was performed with ImaGene 8.0 from BioDiscovery with predetermined settings for spot segmentation and background correction.The background-corrected raw intensity data are logarithmically transformed.Z scores are calculated by subtracting the overall average intensity of all spots within a sample from the raw intensity for each spot, and dividing it by the standard deviations (SD) of all of the measured intensities within each sample (Additional file 26: Table S12). ", "section_name": "Phosphoproteomic screen of U23674", "section_num": null }, { "section_content": "The Probabilistic Target Inhibition Map (PTIM) approach considers that the underlying mechanism for sensitivity to targeted drugs can be represented by a combination of parallel target groups (all parallel targets need to be inhibited to slow or stop tumor proliferation, similar to Boolean ' AND' logic) and series target groups (inhibiting any all targets in any target group will slow or stop tumor proliferation, similar to Boolean 'OR' logic).For estimating the series and parallel targets, we analyze cancer cell response to multi-target single agent drugs with overlapping but distinct target sets.For instance, drugs having the same selective target (such as pelitinib and erlotinib, which are potent inhibitors of the kinase target EGFR) can show different sensitivity in vitro which can be attributed to the biologically relevant side targets of the drugs.Our framework considers primary and secondary drug targets and generates logical groupings of targets (as single-target or multi-target blocks) that best explain chemical screen response data.We now incorporate secondary information to refine PTIM models. ", "section_name": "Probabilistic target inhibition maps", "section_num": null }, { "section_content": "PTIM models are visually represented as circuit models.Each \"block\" in the circuit represents a combination of two or more gene targets that explain sensitivity of a set of single agent compounds.The drug set represented by an individual block is determined by the PTIM objective function and feature selection algorithm [14,16], and depends on the biological data inputs to the PTIM algorithm. PTIM circuits consist of multiple blocks.Generally, only target combinations of one to four targets are considered during PTIM modeling.Blocks of one target (represented as single inhibitor symbol, T 1 ) are called \"single points of failure\", i.e. single targets which alone explain the sensitivity of one or more drug screen agents.Combinations of two targets are visually represented by a rectangular block with two inhibitor symbols (block T 2 -T 3 ).Combinations of three targets are visually represented by a circular block with three inhibitor symbols (block T 4 -T 5 -T 6 ).Combinations of four targets are visually represented by a circular block with four inhibitor symbols (block T 7 -T 8 -T 9 -T 10 ).Each block has an associated score value (e.g.0.825, 0.800, 0.775, 0.750, respectively) that represents the scaled sensitivity of all drug screen agents grouped in the block's target combination [14,16].In brief, all single agent sensitivities (as IC 50 values) are log 10 scaled and converted to [0,1] sensitivity values via the following equation: Thus, the lower the IC 50 , the higher the sensitivity score.The score assigned to each block is a determined by the sensitivity of the drug screen agents assigned to the block following several correction factors [14,16].The shape of blocks in PTIM circuits are meant to serve as a convenient visual representation; ordering of PTIM circuit blocks are determined by overall score, with highest scored blocks on the left descending to lowest scored blocks on the right.The general PTIM algorithm is presented in previously published work [14,[16][17][18].Methods for integration of secondary biological data are provided in the methods sections for modeling of U23674 and modeling of PCB490. ", "section_name": "PTIM circuit models", "section_num": null }, { "section_content": "PTIM circuit models are also designed to visually represent the clinical challenges PTIM modeling seeks to address.Synergistic drug combinations can be selected for any block with two or more targets by selecting two (or more) drugs which inhibit all targets in the block; the selected combination should kill cancer cells while monotherapy treatment would not.For example, based on (block T 2 -T 3 ), a drug inhibiting T 2 and a drug inhibiting T 3 will individually not slow tumor growth for the sample patient, while the combination T 2 + T 3 will. Drug screening multiple spatially-distinct sites from a solid tumor can result in heterogeneous single agent sensitivity.Target group blocks identified as common amongst PTIM models from each distinct region can be used to design a drug combination that should slow or stop tumor growth across the entire heterogeneous tumor.Multi-site PTIM models can thus define heterogeneity-aware drug combinations. Each block in a PTIM circuit represents a set of effective treatment options; effective options on parallel biological pathways represent multiple distinct treatment options which can individually slow tumor growth.A drug combination which inhibits multiple parallel biological pathway blocks can shut down potential survival mechanisms for cancer cells, thus abrogating development of resistance.Series PTIM blocks can thus define resistance abrogating drug combinations. ", "section_name": "Synergy, heterogeneity, and resistance via PTIM models", "section_num": null }, { "section_content": "Probabilistic Target Inhibition Maps (PTIMs) were used for integrative analysis of U23674 biological data [16][17][18]. ", "section_name": "Integrative nonlinear Boolean modeling for U23674", "section_num": null }, { "section_content": "For targets common to both RNA expression data and drug-target interaction data, we use gene expression data to eliminate possible false positives from chemical screen results and to narrow down the true positives among relevant targets identified by the PTIM approach.False positives are defined here as targets that are inhibited by effective drugs but are not expressed in cancer cells at levels above matched normal cells.Note that we consider the effect of a molecularly-targeted drug is to inhibit the target when it is expressed, thus underexpressed drug targets will have limited impact on drug response.Here, over-expression is determined as gene expression in the tumor sample 50% greater than that in the control sample.The RNA-seq target set is used for PTIM creation via the published model development algorithms. Formally, RNA-seq data is integrated as below: ∀ x∉T∩G keep target x for consideration RAPID siRNA screen integration RAPID screen results identify high sensitivity single target mechanisms of cancer cell growth inhibition; identified hit targets were set as \"required\" (forced inclusion) in the RAPID siRNA PTIM model effective as sensitive siRNA targets may explain drug sensitivity of agents inhibiting the siRNA targets.Targets not identified by RAPID screening could still have effect in multi-target combinations, and thus were retained for consideration.The RAPID target set is used for PTIM creation via the published model development algorithms. Formally, RAPID siRNA data is integrated as below: T≔targets inhibited in drug screen G≔targets with RAPID siRNA viability data RAPID x ð Þ≔cell viability following siRNA knockdown of target x μ; σ ð Þ≔mean and standard deviation of RAPID siRNA dataset ", "section_name": "RNA-seq integration", "section_num": null }, { "section_content": "", "section_name": "∀ x∉T∩G keep target x for consideration", "section_num": null }, { "section_content": "The phosphoproteomics screen results identify differentially phosphorylated targets and associated pathways, phosphorylation of these targets may be pushing the system towards a particular phenotype, and intervention in the form of changing phosphorylation status might result in significant changes to the system.S10).Cell plates were then washed in Phosphate Buffered Saline (PBS, Gibco, Grand Island, New York), trypsonized with Trypsin-EDTA (0.25%) (25,200,056, Thermo Fisher Scientific), and screened using the Roche Orphan Kinome Screen (Additional file 11: Figure S11, Additional file 29: Table S15).Rewiring data was used to generate PTIM models to identify post-intervention changes to U23674 survival pathways (Additional file 12: Figure S12, Additional file 27: Table S13). Resistance abrogation experiments for PCB197 and S1-12 PCB197 PPTI screen data and S1-12 PPTI screen data were used to generate PTIM models to identify canine and human cross-species mechanistic targets for undifferentiated pleomorphic sarcoma.Consensus targets were chosen for their appearance in human and canine PTIM models; two drugs (obatoclax, an MCL1 inhibitor and panobinostat, a pan-HDAC inhibitor) that most effectively inhibited PTIM-identified blocks at clinically achievable concentrations were selected for validation.Potential for resistance abrogation by targeting 2 blocks common to both human and canine PTIM models directed a six-arm proof-of-principle experiment to show that inhibition of multiple blocks inhibited could abrogate tumor cell resistance.PCB197 and S1-12 cell cultures were seeded in quadruplicate on 6-well plates (6 plates per cell model) with 10,000 cells per well.Cells were plated 24 h prior to incubation with any drug.The drug concentrations chosen were 1.5 times the EC 50 of the PTIM target of interest.The drug selection was based on desired targets, as well as requiring drug concentration for reaching 1.5 times target K d must also be less than the maximum clinically achievable concentration. One plate per cell model was assigned to each of the 6 treatment arms: 1) vehicle control; 2) obatoclax for 6 days; 3) panobinostat for 6 days; 4) obatoclax for 3 days, wash, then panobinostat for 3 days; 5) panobinostat for 3 days, wash, then obatoclax for 3 days; 6) obatoclax + panobinostat simultaneously for 6 days.After 6 days, culture plates were washed with PBS and fresh DMEM with 10% FBS was placed in each well.Wells were monitored until confluency was observed.The primary study endpoint was days to well confluency as determined by a single user.Cells were also counted manually with a hemocytometer and photographed to confirm consistency of the user's definition of confluency.If after 100 days the cells did not reach confluency, the remaining cells are counted and the study concluded.The experimental design and results are available in Fig. 5. ", "section_name": "Kinexus phosphoproteomics screen integration", "section_num": null }, { "section_content": "We orthotopically engrafted adult SHO (SCID/hairless/outbred) mice (Charles River, Wilmington, Massachusetts) with 10 6 U23674 cells.Engraftment was performed after injuring the right gastrocnemius muscle by cardiotoxin injection as previously described [35].Mice were assigned to treatment arms randomly without a specific assignment strategy.Treatment commenced 2 days after engraftment; mice were treated with vehicle control (tartaric acid + TWEEN80/methylcellulose), 50 mg/kg OSI-906, 150 mg/kg GDC-0941, and combination 50 mg/kg OSI-906 plus 150 mg/kg GDC-0941.Each arm was assigned n = 8 mice per arm.Sample size was selected to provide 90% power for the statistical tests.The GDC-0941 arm lost one mouse during oral gavage; the corresponding data point was censored.Dosing schedule was once daily by oral gavage up to day 5, at which time dosing was performed every other day due to weight loss on day 4.The change in dosing schedule stabilized weight loss.The endpoint considered for the study and survival analysis was tumor volume = 1.4 cc.All drug studies in mice were performed after receiving approval from the IACUC at Oregon Health and Science University.Variances between compared groups were similar per Greenwood's Formula.No blinding was performed during in vivo experiments.No adverse events were noted.All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the Oregon Health & Science University.At conclusion of the study, mice were sacrificed via isoflurane overdose followed by cervical dislocation. ", "section_name": "Orthotopic allograft studies for U23674", "section_num": null }, { "section_content": "Adult female stock mice (Envigo Foxn1 nu Athymic nudes) were implanted bilaterally with approximately 5x5x5mm fragments subcutaneously in the left and right flanks with JAX PDX model of Human Epithelioid Sarcoma (J000078604 (PCB490) -JAX-001).After the tumors reached 1-1.5 cm 3 , they were harvested and the viable tumor fragments approximately 5x5x5 mm were implanted subcutaneously in the left flank of the female study mice (Envigo Foxn1 nu Athymic nudes).Each animal was implanted with a specific passage lot and documented.J000078604 (PCB490) -JAX-001) was P4.Tumor growth was monitored twice a week using digital calipers and the tumor volume (TV) was calculated using the formula (0.52 × [length × width 2 ]).When the TV reached approximately 150-250 mm 3 animals were matched by tumor size and assigned into control or treatment groups (3/group for J000078604 (PCB490) -JAX-001).Dosing was initiated on Day 0. After the initiation of dosing, animals were weighed using a digital scale and TV was measured twice per week.For J000078604 (PCB490) -JAX-001, sunitinib (reconstituted in 5% DMSO + corn oil) was administered PO QD for 21 days at 30.0 mg/kg/dose and BEZ235 (reconstituted in 10% N-Methyl-2-pyrrolidone [NMP] + 90% polyethylene glycol 300) was administered PO QD for 21 days at 25.0 mg/kg/ dose alone and in combination.No adverse events were noted.At conclusion of the study, mice were sacrificed via isoflurane overdose followed by cervical dislocation. ", "section_name": "Patient derived xenograft (PDX) model testing for PCB490", "section_num": null }, { "section_content": "Spearman correlation coefficients for Epithelioid sarcoma drug screen response data were calculated in SAS, correlating drug screen IC 50 values between all samples.Statistical comparison of correlation coefficients between separate groups was performed in SAS using two-tailed student's T-test. The Kaplan-Meier curves for the U23674 in vivo orthotropic allograft studies were generated and compared with logrank statistical tests.No blinding was performed.Analysis was performed by an external group of statisticians (MWG, BH, JM, SG). P-values for the PCB490 PDX experiment were generated using a repeated measures linear model of tumor size in terms of group, time, and the group by time interaction based on an autoregressive order 1 correlation assumption with SAS Version 9.4 for Windows (SAS Institute, Cary, NC).Analysis was performed by an external group of statisticians (MWG, BH, JM). ", "section_name": "Statistics", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "The key PTIM modeling assumption is that in vitro drug sensitivity in cancer cells is driven by a small subset of key gene targets uniquely determined by the patient's biology, and that patient-specific drug sensitivity is most accurately predicted by multivariate modeling of autologous drug sensitivity data.The PTIM pipeline requires drug screening data from multiple (60+) monotherapy agents with quantified drug-target EC 50 values (Fig. 1, Testing Step).PTIM modeling specifically takes advantage of the promiscuity of targeted compounds by incorporating main-target and off-target EC 50 values during modeling.Correspondingly, PTIM models will better represent the underlying biology of individual cancer samples when complete drug-target interaction EC 50 information is available.Integration of additional patient-specific molecular data (e.g., exome-seq, RNA-seq, phosphoproteomics, siRNA-mediated gene knockdown, Fig. 1,Testing Step) identifies targets of interest to further refine target selection during model creation. Drug sensitivity data and secondary molecular data are provided as inputs to the PTIM computational framework [14][15][16][17][18][19], which provides as output a mathematical model quantifying expected sensitivity of multi-target inhibition of the patient's cancer cells.The model approaches sensitivity prediction as a feature selection machine learning problem, where the \"features\" are the gene targets inhibited by individual drugs.The objective of the PTIM analysis approach is to find feature sets which group sensitive and insensitive drugs together into binary \"bins\", representing a set of inhibited targets.A feature set where drugs in the same bin have similar sensitivity values is considered more optimal than a feature set where bins have high variance.The addition of molecular sequencing data can eliminate certain features from consideration if they are absent in the tumor (e.g.no expression of the gene per RNA-seq data) or can increase likelihood of a feature being included in the model if the feature is of high interest (e.g.highly expressed per RNA-seq, or mutated per exome-seq).The full details of integration of molecular is available in the methods section, including a detailed description of integration of molecular data to drug screening data for validation experiments presented in this manuscript. Multi-target sensitivity mechanisms are represented graphically as \"tumor cell survival circuits\" (Fig. 1,Modeling Step) where target combinations are denoted as \"blocks\" (e.g. Figure 1, Modeling Step inhibitor symbols A, B, C + D).The value in the center of each PTIM block represents expected scaled sensitivity following inhibition of associated block targets.The resulting PTIM model enables combination therapy assignment via matching of targets in high-sensitivity PTIM blocks to drugs in clinical investigation or clinical use.A single block denotes monotherapy (e.g.A, B) or combination therapy (synergistic targets, e.g.C + D), while multiple blocks represent independent treatments which can be leveraged to abrogate cancer cell resistance. If PTIM models from spatially-distinct tumor sites are available, consensus therapy can be selected from distinct models to mitigate potential intra-tumor heterogeneity.When available, additional patient tumor tissue can be used to validate PTIM-predicted combination therapy in vitro or in vivo (Fig. 1,Validation Step).PTIM modeling is the foundation of our personalized therapy pipeline built with the goal to address the unmet clinical needs of the 600,000 patients dying from cancer every year [1]. The MATLAB package to generate basic PTIM models was published in conjunction with a previous publication [16] and is available online (http://www.myweb.ttu.edu/rpal/Softwares/PTIM1.zip). ", "section_name": "Computational analysis of functional and molecular data via PTIM analysis", "section_num": null }, { "section_content": "For our 2-drug synergy proof-of-concept study, we used a low passage primary tumor cell culture of a GEMM-origin aRMS tumor designated U23674 [36] as a pilot study of the PTIM personalized therapy pipeline.From our previous work [35,37] we reasoned that kinases would be fundamental to the biology of aRMS, thus we interrogated U23674 drug sensitivity via three kinase inhibitor compound libraries: the GlaxoSmithKline (GSK) Open Science Orphan Kinome Library (GSK screen), the Roche Orphan Kinome Screen Library (Roche screen), and a custom Pediatric Preclinical Testing Initiative Drug Screen Version 2.1 (PPTI screen). The GSK screen [38] consists of 305 compounds with experimentally quantified drug-target interaction EC 50 values.Of the 305 screened compounds, 40 (13%) caused at least 50% cell growth inhibition at or below maximum tested in vitro dosage in U23674, hereafter defined as a compound \"hit\" (Additional file 1: Figure S1 and Additional files 15 and 16: Tables S1 andS2).The Roche screen consists of 223 novel kinase inhibitor compounds, most with quantified drug-target interactions; 21 of 223 compounds (9.4%) were hits on U23674 (Additional file 2: Figure S2 and Additional files 17, 18 and 19: Tables S3, S4 andS5).The PPTI screen consists of 60 preclinical-or clinical-stage targeted agents; 28 of 60 compounds (46.7%) were hits on U23674 (Additional file 3: Figure S3 and Additional files 20 and 21: Tables S6 andS7). Additionally, U23674 primary tissue was sequenced to enhance therapy design (tumor whole exome sequencing, matched normal whole exome sequencing, and whole transcriptome sequencing, Additional files 22 and 23: Tables S8 and S9).Exome sequencing of U23674 did not identify any druggable targets both mutated and amplified (Additional file 4: Figure S4 and Additional files 22 and 23: Tables S8 andS9); six genes possessed activating mutations (Fat4, Gm156, Mtmr14, Pcdhb8, Trpm7, Ttn, Zfp58) and one gene possessed a high-impact frameshift indel (Ppp2r5a); none of these seven gene targets are druggable.No gene with a mutation or indel is druggable.Four druggable gene targets show evidence of copy number gain (Gsk3a, Epha7, Psmb8, Tlk2).Gsk3a, Psmb8, and Tlk2 all show neutral expression or underexpression by RNA-seq.Gsk3a inhibitors were effective in 12 of 72 inhibitors (16.667%) across three screens, suggesting Gsk3a is not critical for cancer cell survival in U23674.Psmb8 inhibition showed in vitro efficacy in nearly all tested cell cultures across multiple tumor types (unpublished internal data) and, along with lack of overexpression, was thus treated as an in vitro screening artifact; furthermore, clinical response of solid tumors to proteasome inhibitors has been limited [39].Tlk2 has no published inhibitor compounds.While overexpressed, the Epha7 inhibitor on the PPTI drug screen was ineffective against U23674.Therapy assignment via exome sequencing alone would thus have limited clinical utility for U23674. ", "section_name": "Proof-of-concept of synergy prediction by PTIM modeling Chemical screening, biological interrogation, and PTIM modeling of a genetically engineered mouse model (GEMM)-origin aRMS", "section_num": null }, { "section_content": "The high average level of target coverage (24 compounds/ target), the inclusion of both typical and atypical kinase target combinations, and the thorough characterization of drug-target interactions made the GSK screen the most complete dataset available and was thus selected to guide in vitro and in vivo validation experiments.Baseline (chemical screen data only), RNA-seq informed-, exome-seq informed, siRNA interference informed-, and phosphoproteomics informed-PTIM models were generated from the GSK screen data (Fig. 2a-c, Additional file 5: Figure S5, Additional files 24, 25, 26, 27: Tables S10-S13).PTIM-identified targets were consistent with known targets of interest in aRMS [40,41] and identified gene targets involved in established protein-protein interactions [42] (Additional file 6: Figure S6).As multi-drug combinations impart toxicity concerns and dosing limitations, we focus on PTIM blocks (combinations of two or more targets) treatable by at most two drugs.Baseline and genomics-informed PTIM models were also generated for the PPTI and Roche screens (Additional file 7: Figure S7, Additional file 27: Table S13), however no validation experiments based on PPTI or Roche PTIM models were performed due to focus on the GSK screen results. We selected two combinations for in vitro synergy validation: 1) the RNA-seq-informed target combination Igf1r & Pik3ca (Fig. 2b) with combination therapy OSI-906 + GDC-0941 (a Pik3ca inhibitor selective against Akt/mTOR), and 2) The baseline target combination Igf1r & Insr & Pka with combination therapy OSI-906 (an Igf1r and Insr inhibitor) + SB-772077-B (Pka inhibitor, denoted GSK-PKA in figures).All compounds were selected based solely on selectivity of interaction with the PTIM-identified targets. We selected the RNA-seq-informed drug combination due to high block sensitivity, targetability by a two-drug combination, and our previous work showing higher correlation between transcriptome status and drug sensitivity Fig. 2 (See legend on next page.)[14].The baseline combination was selected due to targetability by a two-drug combination, higher score compared to other two-drug options, and to serve as a comparison between baseline PTIM models and molecularly-informed models.In vitro validation experiments for OSI-906 + GDC-0941 (Fig. 2d-e) demonstrated synergy as determined by non-constant ratio Combination Index [43] (CI) values (Additional file 28: Table S14).Low-dose combination experiments were also performed to confirm PTIM-predicted drug mechanism of action (Additional file 8: Figure S8, Additional file 28: Table S14).Both full-dose and low-dose OSI-906 + SB-772077-B in vitro validation experiments (Additional file 9: Figure S9) demonstrated non-constant ratio Combination Index synergy (Additional file 28: Table S14), though overall cell viability of was OSI-906 + SB-772077-B than higher the RNA-seq-informed combination.In vitro results support the potential of baseline and molecularly-informed PTIM modeling to discover synergistic target combinations, though inclusion of molecular data may narrow focus on targets which are overexpressed and/or aberrant and thus more likely to respond to drug treatment. ", "section_name": "Probabilistic target inhibition map (PTIM) modeling identifies 2-drug combinations with synergy in vitro", "section_num": null }, { "section_content": "To explore tumor rewiring (activation of secondary signaling pathways to improve chance of survival) following synergy-focused intervention, we treated U23674 cell populations with low-dose monotherapy or combination therapies defined in initial in vitro validation experiments, and subsequently screened the populations via the Roche screen (Additional files 10 and 11: Figures S10 andS11 and Additional file 29: Table S15).Unsurprisingly, the cell populations showed evidence of rewiring within hours of monotherapy or combination therapy intervention (Additional file 12: Figure S12, Additional files 27 and 28: Tables S13 andS14), emphasizing the importance of simultaneous, multi-pathway drug combinations at full therapeutic doses.While PTIM modeling currently focuses on 2-drug combinations to minimize toxicity concerns, PTIM-predicted combinations of three or more drugs are possible with sufficient evidence of safety and efficacy. ", "section_name": "Tumor cell rewiring following synergy-focused combination therapy", "section_num": null }, { "section_content": "Having demonstrated in vitro synergy, we next validated OSI-906 + GDC-0941 in vivo.We designed a four-arm orthotopic allograft study (Fig. 2f) comparing vehicle, OSI-906 (50 mg/kg), GDC-0941 (150 mg/kg), and OSI-906 (50 mg/kg) + GDC-0941 (150 mg/kg).Kaplan-Meier survival analysis (Fig. 2g) showed improvement in mouse lifespan from combination treatment (under Bonferroni correction: Vehicle -Combo, p = 0.005, OSI-906 -Combo, p = 0.014, GDC-0941 -Combo, p = 0.079.In all cases, p < 0.05 uncorrected).Survival of mice treated with either OSI-906 or GDC-0941 alone was indistinguishable from treatment by vehicle (p > 0.5, both corrected and uncorrected).Since a PTIM block represents targets which are weak independently but synergistic together, U23674 in vivo data supports the hypothesis underlying our modeling approach: synergistic combination targets can be identified through computational modeling of monotherapy chemical agents. ", "section_name": "Probabilistic target inhibition map (PTIM) modeling predicts 2-drug combination with in vivo efficacy", "section_num": null }, { "section_content": "EPS is a soft tissue sarcoma of children and adults for which chemotherapy and radiation provides little improvement in survival [44].Effective options beyond wide surgical excision are presently undefined [45], making EPS a viable test case for developing targeted personalized therapies. We have developed several new heterogeneous EPS preclinical resources: three new unpublished cell cultures, as well as (to our knowledge) the first reported patient-derived xenograft (PDX) model of EPS derived from a 22-year-old female with a large proximal (shoulder) EPS tumor (Fig. 3a).The tumor sample was obtained from surgical resection and was assigned the internal identifier PCB490.Due to the size of the acquired tumor sample and the potential [16] when the targets are inhibited via one or more chemical compounds.More information can be found in prior publications [16,18].In (d-e), results are based on n = 3 technical replicates with n = 4 replicates per treatment condition.d Dose response curve for OSI-906 varied dosage + GDC-0941 fixed dosage.The response for GDC-0941 at varied dosages is included.e Dose response curve for GDC-0941 varied dosage + OSI-906 fixed dosage.The response for OSI-906 at varied dosages is included.f Schematic representation of in vivo experiment design.g Kaplan-Meier survival curves for in vivo orthotropic mouse experiment.Mice were treated with vehicle (n = 8 mice, black line), 50 mg/kg OSI-906 (n = 8 mice, blue line), 150 mg/kg GDC-0941 (n = 7 mice, red line), or combination 50 mg/kg OSI-906 + 150 mg/kg GDC-0941 (n = 8 mice, purple line).The medicine bottle image is public domain, provided by user Kim via clker.com(http://www.clker.com/clipart-blank-pill-bottle-3.html) for heterogeneity in solid tumors [46], we divided the ~3 cm 2 resected tumor mass into five spatially-distinct regions (designated PCB490-1 through PCB490-5) and cultured each region to develop heterogeneous cell models (Fig. 3a).PCB490 cultures were maintained at low passage to minimize biological drift from the original patient samples.To confirm EPS diagnosis, three of five tumor sites (1, 2, and 5) were validated by western blot for INI1 protein, which is absent in 93% of EPS samples (Fig. 3b) [44] as well as in published cell lines [21,22].Multiple sites were submitted to The Jackson Laboratory for establishment of PDX models; PCB490-5 PDX developed a passageable tumor that matched the original PCB490-5 sample by both histology and INI1 immunohistochemical staining (Fig. 3c-f ). ", "section_name": "Proof-of-concept of heterogeneity-consensus 2-drug combinations predicted by PTIM modeling Development of heterogeneous cell models of Epithelioid Sarcoma (EPS)", "section_num": null }, { "section_content": "Cell cultures PCB490-3, PCB490-4, and PCB490-5 grew to sufficient populations (minimum 3 × 10 6 cells) at low passage (passage 2 or below) to allow for drug screening via the investigator-selected 60-agent screen denoted Drug Screen V3 (Fig. 3g, Additional file 30: Table S16) and the previously described Roche screen (Additional file 31: Table S17).Drug screen endpoints were per-drug IC 50 values.PCB490 primary tissue was sequenced for tumor whole exome sequencing, matched normal whole exome sequencing, and whole transcriptome sequencing (Additional file 13: Figure S13, Additional files 32 and 33: Tables S18 and S19).Sequencing identified germline and tumor amplified, expressed, high-impact druggable variants in two genes (ABL1, NOTCH1) and expressed, medium impact variants in three additional genes (MDM4, PAK4, MAP4K5).All five variants were identified in both tumor and normal (germline) samples.The ABL1 variant was previously identified in the 1000 Genomes Project [47].The ABL1, NOTCH1, MDM4 and PAK4 variants were previously submitted to the dbSNP database [48].All variants are of unknown clinical significance (Additional file 34: Table S20) [48,49].PCB490 drug screening results revealed no pathwayspecific drug sensitivity of mutated genes (Additional file 14: Figure S14) suggesting therapy assignment via exome sequencing alone would likely have limited clinical utility for PCB490. To compare drug sensitivity of PCB490 with other EPS models, three cell lines (ESX, FU-EPS-1, and VA-ES-BJ), a second human-derived cell culture (PCB495), and the SkMc skeletal myoblast cell line were assayed with Drug Screen V3 (Fig. 3g, Additional file 35: Table S21).Drug Screen V3 responses were compared by calculating Spearman correlation coefficients (Fig. 3h) to quantify the similarity between the new EPS models and existing EPS cell models.For the purpose of this analysis, we treat the PCB490 cultures from different regions as independent samples.Correlation within primary cell cultures (PCB490 sites and PCB495) was significantly higher than correlation between primary cultures and cell lines (μ = 0.6466 vs. μ = 0.4708, p < 0.01), suggesting EPS primary cultures may be biologically distinct from EPS cell lines.PCB490 drug screen response differed between sample locations millimeters away from each other, reflective of biological differences arising from spatial tumor heterogeneity.Nonetheless, correlation between chemical screen results from PCB490 cultures was significantly higher than correlation between PCB490 cultures and PCB495 cultures/ EPS cell lines (μ = 0.7671 vs. μ = 0.4601, p < 0.001), suggesting that treatments for PCB490 may be better defined solely by PCB490 biological data. ", "section_name": "Drug screening, sequencing, and comparison of heterogeneous EPS cell cultures", "section_num": null }, { "section_content": "Highly correlated yet heterogeneous PCB490 drug sensitivity data guided us towards PTIM modeling to design a heterogeneity-consensus personalized drug combination therapy for PCB490.PTIM models of PCB490-3 (Fig. 4a, Additional file 27: Table S13), PCB490-4 (Fig. 4b, Additional file 27: Table S13), and PCB490-5 with integrated RNA-seq data (Fig. 4c, Additional file 27: Table S13) indicated common efficacious mechanisms across the heterogeneous tumor sites: epigenetic modifiers (HDAC, EHMT), PI3K/mTOR inhibition, and VEGF (KDR) signaling inhibition.We focused on high-scoring PTIM blocks treatable by a two-drug combination, resulting in selection of BEZ235 (PI3K/mTOR inhibitor) and sunitinib (poly-kinase inhibitor, including KDR and AXL).BEZ235 + sunitinib was selected solely based on PTIM modeling data, agnostic to previous use of sunitinib in EPS [50]. To replicate potential clinical conditions for personalized combination therapy, we bypassed in vitro validation and directly initiated in vivo testing of BEZ235 + sunitinib in the PCB490 PDX model.Though the PCB490 PDX model originates from the PCB490-5 region, heterogeneity of PCB490 suggests the tumor section used to establish the PCB490 PDX can be considered a unique heterogeneous region.PDX testing of BEZ235 + sunitinib demonstrated significant slowing of tumor growth over vehicle control (92% slower tumor growth at Day 19, p = 0.01) (Fig. 4d).In statistical analysis restricted to treated animals at Day 19, BEZ235 + sunitinib significantly slowed PDX tumor growth compared to both BEZ235 (p = 0.01) and sunitinib (p = 0.01) alone (Fig. 4d).Proof-of-concept of resistance-abrogating 2-drug combinations predicted by PTIM modeling PTIM modeling of undifferentiated pleomorphic sarcoma (UPS) samples guides cross-species resistance-abrogating drug combination in vitro The previously discussed U23674 rewiring experiment emphasized the need for multi-pathway targeting when developing personalized treatments.The PTIM modeling approach identifies mechanisms driving in vitro drug sensitivity by identifying effective target combination \"blocks\"; two blocks operating on different biological pathways represent two independent treatment mechanisms.We reasoned that two-block inhibition could result in resistance-abrogating combination treatments, thus we validate a drug combination designed from two PTIM blocks representing independent biological pathways.PTIM modeling of PPTI screen data from a UPS derived from a 75-year-old man (PCB197, Fig. 5a, Additional file 36: Tables S22) and a canine-origin UPS (S1-12, Fig. 5b, Additional file 36: Table S22) identified species-consensus drug sensitivity mechanisms targetable by a 2-block, 2-drug combination (Fig. 5c,d,Additional file 27: Table S13): panobinostat (pan-HDAC inhibitor, HDAC7 block) and obatoclax (MCL1 inhibitor).The combination of panobinostat + obatoclax was predicted to abrogate resistance mechanisms and prevent cancer cell rewiring and regrowth; furthermore, the cross-species nature of the experiment supports the resistance-abrogation effect not being model specific. To validate in vitro resistance abrogation across species, we performed identical six-arm in vitro trials for PCB197 and S1-12.Each arm represented a different combination method for the cross-species combination: vehicle treatment, monotherapy treatment, serial monotherapy treatment (panobinostat then obatoclax, obatoclax then panobinostat), and simultaneous combination treatment (concurrent panobinostat + obatoclax) (Fig. 5e).Resistance abrogation in each arm was determined by cellular regrowth over 100 days following treatment.Rewiring and regrowth was expected for all monotherapy and serial treatment modalities.All arms except the simultaneous combination treatment arm experienced cellular regrowth, indicating the development of resistance.In both cultures, the simultaneous combination treatment arm showed no cellular regrowth over 100 days, indicating the combination potentially addressed resistance mechanisms (Fig. 5f,g). ", "section_name": "PTIM modeling guides heterogeneity-consensus in vivo drug combination", "section_num": null }, { "section_content": "The work presented here represents validation experiments for three aspects of PTIM-guided personalized cancer therapy design: drug sensitivity and synergy prediction in a GEMM-origin aRMS, heterogeneity-consensus drug combination design and validation in the first-reported EPS PDX model, and mitigation of cancer cell resistance mechanisms in cross-species in vitro validation experiments.Our studies suggest the high value of combining functional screening data with secondary molecular data (especially RNA-seq data) in the design of personalized drug combinations as a supplement to or alternative to DNA sequencing-based therapy assignment.While increased effort is required to generate functional data, the additional information and evidence may prove useful in designing therapeutically effective personalized cancer treatments. Critically, the timeframe for PTIM-based combination therapy design is less than the time required for standard high-throughput sequencing experiments.The PTIM analysis pipeline can be performed in under 2 weeks and without the explicit need for sequencing results.Currently, the time limiting step in integrative PTIM analysis is exome and RNA sequencing, for which new technology is rapidly reducing time and monetary cost.Functional drug screening in standard well plates can be performed for under $300, and CLIA-certified physical sequencing experiments are now under $500 per analyte per experiment; the cost of a complete functional and molecular analysis now represents a fraction of drug cost and may be accessible to a large population of cancer patients. The three PTIM-guided validation experiments serve as proofs-of-concept rather than a full clinical validation.The current study lacks the large sample size necessary to reach definite conclusions on the large-scale efficacy of PTIM-based personalized cancer therapy.Any treatment strategy, especially a personalized approach, requires a large population to draw clinically-relevant conclusions.Increasing the sample size of personalized treatments designed by the PTIM approach is required to demonstrate clinical use.To that end, the critical next stage in PTIM-based personalized therapy design will be prospective evaluation by partnering with physicians and veterinarians to pilot testing of n-of-1 personalized therapies in individual human patients and animals with spontaneous cancer.As the cost of analysis is low, the major challenges will be 1) administration of FDA-approved drugs, very likely as off-label therapy in combinations potentially not validated in Phase I trials, and 2) financial costs associated with modern targeted therapy regimens, which may currently be prohibitive for some patients. As drug screen results ultimately guide PTIM modeling, computational modeling of different disease types will require designing disease-specific compound screens to maximize the breadth and depth of disease-relevant multi-target interactions [15].Similarly, different types of secondary molecular data influences target selection during PTIM model construction depending on the underlying analyte or perturbation, with different secondary datasets expectedly producing different PTIM models.Selection of secondary datasets to generate for individual cases will depend on availability of tumor tissue and expected predictive utility of individual datasets.Based on widespread clinical utility and published studies, the current standard secondary datasets for PTIM modeling are exome sequencing data and RNA sequencing data [14].As high-throughput analysis of additional biological analytes becomes available through CLIA certified procedures, new datatypes will be integrated into PTIM models.In particular, recent advances in robust generation of proteomics data from patient samples [51][52][53] may enable routine integration of proteomics data into PTIM modeling beyond the test case presented in this work. PTIM-based personalized cancer therapy also requires development of personalized toxicity and dosing prediction methods for designing maximally effective, minimally toxic drug combinations.Research on toxicity prediction is underway, as is research on incorporating chemotherapy backbone into drug combination predictions.While validated PTIM models are currently based on low-passage cell cultures (U23674, PCB490, S1-12, PCB197), future application of PTIM models will use direct-to-plate tumor screening to best recapitulate the patient's disease state and to remove the dependence on cell culture establishment.Finally, we will pursue expansion of disease-consensus PTIM modeling [19] to establish new disease-specific drug combinations based on integrated drug screening and high-throughput sequencing data. ", "section_name": "Discussion", "section_num": null }, { "section_content": "PTIM-based personalized combination therapy has been designed to uniquely leverage patient-specific functional and biological data to address some of the critical unmet clinical needs of the 60% of cancer patients for whom tumor DNA analysis is uninformative [2] and the 600,000 patients lost to cancer every year [1] who have exhausted clinical options.PTIM modeling can also meet the needs of cancer patients with rare diseases, such as the spectrum of 60+ cancers known as non-rhabdomyosarcoma soft tissue sarcomas (including EPS) for which effective clinical treatments may not exist and may never be developed due to a paucity of disease models for research.These two groups represent a significant cancer patient population for which PTIM modeling may provide evidence-based treatment options where no therapeutic avenues exist. compounds inhibiting mutated and expressed targets in PCB490.Red in the IC 50 and EC 50 tables indicates low IC 50 and EC 50 values, respectively.No single target or combination of targets showed uniform efficacy across all PCB490 cultures, suggesting variations alone or in conjunction with transcriptome sequencing would not have identified actionable therapeutic targets.Heat values in the IC 50 section of the table represent drug sensitivities as IC 50 values, between 1 nM (red) and 6 μM or above (white).Heat values in the EC 50 section of the table represent quantified drug-target interaction between chemical agents and gene targets, quantified as 50% inhibitory concentrations between 1 nM (red) and 6 μM or above (white), with grey representing no interaction.(TIF 13895 kb) ", "section_name": "Conclusion", "section_num": null } ]	[ { "section_content": "We thank William Zuercher and David Drewry at GlaxoSmithKline and Paul Gillespie at Roche for making the respective compound libraries available for the community and this study.Funding This work was supported by the Scott Carter Foundation Fellowship grant (to N.E.B), the SuperSam Foundation Fellowship grant (to N.E.B), the Prayers for Elijah Foundation (to C.K. and N.E.B.), NSF award CCF0953366 (to R.P.), the Damon Runyon-Sohn & St. Baldrick's Foundation training grants (to L.E.D.), the AAO-HNSF Saidee Keller Memorial Resident Research Grant, American Academy of Otolaryngology -Head & Neck Surgery Foundation (AAO-HNSF) and the Centralized Otolaryngology Research Effort (CORE) Study Section (to M.N.G), as well as by a gift from an anonymous donor.None of the funding bodies were involved in design or analysis of the experiments, or interpretation of the results. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "All analyzed data is available in the supplemental materials for this manuscript.RNA sequencing data is available through the Gene Expression Omnibus (GEO, GSE128766), and DNA sequencing data is available through the European Genome-Phenome Archive (EGA, EGAD00001004885). ", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "Additional file 1: Figure S1.Heat map of merged chemical screen, RNA-seq, siRNA, and phosphoproteomics results for GlaxoSmithKline (GSK) Orphan Kinome screen.Due to the large number of compounds and protein targets, only a limited scope of compounds and targets is shown here (for full data, see Additional file 15: Table S1).Bright red indicates high sensitivity values, gradating down to white meaning low sensitivity.Gray indicates no interaction or no available data.Asterisk indicates targets later validated in vivo.(TIF 38030 kb) Additional file 2: Figure S2.Heat map of merged Roche Orphan Kinome chemical screen, RNA-seq, siRNA, and phosphoproteomics results.Due to the large number of compounds and protein targets, only a limited scope of compounds and targets is shown here (For full data, see Additional file 17: Table S3).Bright red indicates high sensitivity values, gradating down to white meaning low sensitivity.Gray indicates no interaction or no available data.(TIF 58505 kb) Additional file 3: Figure S3.Heat map of joint version 2.1 chemical screen, RNA-seq, siRNA, and phosphoproteomics results.Due to the large number of compounds and protein targets, only a limited scope of compounds and targets is shown here (For full data, see Additional file 21: Table S7).Bright red indicates high sensitivity values, gradating down to white meaning low sensitivity.Gray indicates no interaction or no available data (TIF 57387 kb) S1 Authors' contributions NEB performed computational modeling and analysis, designed and analyzed experiments, and oversaw the study.RP oversaw computational modeling and analysis and oversaw the study.CK designed and analyzed experiments, wrote the manuscript, and oversaw the study.NEB, RR, JA, and CK helped write the manuscript.RR provided additional support for the study.KLM assisted with figure development for the manuscript.Biological resources (cell lines) for in vitro experiments were provided by JNoujaim, YCD, KT, TT, JNishio, RGM, RLJ, MMilovancev provided biological resources.EH prepared drug screen plates with which LED performed drug screening experiments on cell line models.MNG and MNS processed and cultured tumor tissue and performed drug screening experiments on primary cell cultures of fresh tumor tissue.AM performed histological analysis of tissue samples from fresh tumor tissue.MQ performed sequencing data analysis.In-house in vivo experiments were performed by JA.The Champions Oncology mouse studies were overseen by EW and MMancini.The Jackson Laboratory mouse studies were overseen by SA and CB oversaw.RGE performed histological analysis of PDX implanted mice on behalf of the Jackson Laboratory.JEM, MWG, BSH and SG performed statistical analysis and interpretation of in vivo experimental results.All authors read and approved the final manuscript. Human cell lines used in blotting and drug screening experiments did not require ethics approval for usage.All animal procedures performed at Oregon Health & Science University were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the Oregon Health & Science University.All animal procedures performed at The Jackson Laboratory were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at The Jackson Laboratory.All animal procedures performed at Champions Oncology were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Champions Oncology.All human tissue samples were acquired through the Childhood Cancer Registry for Familial and Sporadic Tumors (CCuRe-FAST) tumor banking program.All patients enrolled in CCuRe-FAST provided informed consent via written consent. All aspects of the study were reviewed and approved by the Oregon Health & Science University (OHSU) Institutional Review Board (IRB).Patient data and clinical and pathologic information are maintained in a de-identified database. Competing interests Investigators N.E.B., C.K. and R.P. have previously filed invention disclosures for the probabilistic Boolean model that integrates chemical screening and genomics data, and are in the process of forming a related company.The 's have declared these conflicts to their respective institutions, which are developing conflict of interest management plans.All other authors declare no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Additional files", "section_num": null }, { "section_content": "Additional file 1: Figure S1.Heat map of merged chemical screen, RNA-seq, siRNA, and phosphoproteomics results for GlaxoSmithKline (GSK) Orphan Kinome screen.Due to the large number of compounds and protein targets, only a limited scope of compounds and targets is shown here (for full data, see Additional file 15: Table S1).Bright red indicates high sensitivity values, gradating down to white meaning low sensitivity.Gray indicates no interaction or no available data.Asterisk indicates targets later validated in vivo.(TIF 38030 kb) Additional file 2: Figure S2.Heat map of merged Roche Orphan Kinome chemical screen, RNA-seq, siRNA, and phosphoproteomics results.Due to the large number of compounds and protein targets, only a limited scope of compounds and targets is shown here (For full data, see Additional file 17: Table S3).Bright red indicates high sensitivity values, gradating down to white meaning low sensitivity.Gray indicates no interaction or no available data.(TIF 58505 kb) Additional file 3: Figure S3.Heat map of joint version 2.1 chemical screen, RNA-seq, siRNA, and phosphoproteomics results.Due to the large number of compounds and protein targets, only a limited scope of compounds and targets is shown here (For full data, see Additional file 21: Table S7).Bright red indicates high sensitivity values, gradating down to white meaning low sensitivity.Gray indicates no interaction or no available data (TIF 57387 kb) S1 Authors' contributions NEB performed computational modeling and analysis, designed and analyzed experiments, and oversaw the study.RP oversaw computational modeling and analysis and oversaw the study.CK designed and analyzed experiments, wrote the manuscript, and oversaw the study.NEB, RR, JA, and CK helped write the manuscript.RR provided additional support for the study.KLM assisted with figure development for the manuscript.Biological resources (cell lines) for in vitro experiments were provided by JNoujaim, YCD, KT, TT, JNishio, RGM, RLJ, MMilovancev provided biological resources.EH prepared drug screen plates with which LED performed drug screening experiments on cell line models.MNG and MNS processed and cultured tumor tissue and performed drug screening experiments on primary cell cultures of fresh tumor tissue.AM performed histological analysis of tissue samples from fresh tumor tissue.MQ performed sequencing data analysis.In-house in vivo experiments were performed by JA.The Champions Oncology mouse studies were overseen by EW and MMancini.The Jackson Laboratory mouse studies were overseen by SA and CB oversaw.RGE performed histological analysis of PDX implanted mice on behalf of the Jackson Laboratory.JEM, MWG, BSH and SG performed statistical analysis and interpretation of in vivo experimental results.All authors read and approved the final manuscript. ", "section_name": "Additional files", "section_num": null }, { "section_content": "Human cell lines used in blotting and drug screening experiments did not require ethics approval for usage.All animal procedures performed at Oregon Health & Science University were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the Oregon Health & Science University.All animal procedures performed at The Jackson Laboratory were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at The Jackson Laboratory.All animal procedures performed at Champions Oncology were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Champions Oncology.All human tissue samples were acquired through the Childhood Cancer Registry for Familial and Sporadic Tumors (CCuRe-FAST) tumor banking program.All patients enrolled in CCuRe-FAST provided informed consent via written consent. All aspects of the study were reviewed and approved by the Oregon Health & Science University (OHSU) Institutional Review Board (IRB).Patient data and clinical and pathologic information are maintained in a de-identified database. ", "section_name": "Ethics approval and consent to participate", "section_num": null }, { "section_content": "Competing interests Investigators N.E.B., C.K. and R.P. have previously filed invention disclosures for the probabilistic Boolean model that integrates chemical screening and genomics data, and are in the process of forming a related company.The 's have declared these conflicts to their respective institutions, which are developing conflict of interest management plans.All other authors declare no competing interests. ", "section_name": "Consent for publication Not applicable.", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.1038/s41375-023-01845-9	Clinical impact of TP53 disruption in chronic lymphocytic leukemia patients treated with ibrutinib: a campus CLL study	NA	[ { "section_content": "TO THE EDITOR Disruption of the TP53 gene, either by deletion at chromosome 17p13.1 (del17p) or mutations, is the most important prognostic/ predictive biomarker in chronic lymphocytic leukemia (CLL), also in the context of the novel target therapies including ibrutinib [1][2][3][4].Although TP53 deletion and mutations mostly co-occur and are considered as equal prognosticators, the prognostic value of isolated or concomitant mutations and deletions remains unclear [2,3].Here we applied an ultra-deep next-generation sequencing (NGS) approach in CLL patients treated with ibrutinib, to investigate the clinical impact of TP53 mutations and del17p, either concomitant or isolated, or in relation to their disruption burden. This study, generated in the framework of an institutional Italian multicenter working group on CLL (\"Campus CLL\"), is a retrospective/multicenter analysis of 229 CLL patients treated with ibrutinib in the current clinical practice.All cases have been either referred to a single institution for molecular and cytogenetic analyses (February 2014-February 2021), or retrospectively referred by delivering frozen cell samples taken prior to starting ibrutinib treatment.Clinical outcome data were updated as of October 2021.Eighty patients, included in a previous study [3], are presented here with an updated median follow-up (24.7 months).As a stringent criterion, only patients assayed for TP53 mutation and 17p deletion in the same blood sample taken within 6 months prior to the start of ibrutinib were included.Median follow-up from ibrutinib treatment was 36.3 months (95% CI 29.5-41.5 months); 51 patients were treatment naïve (TN) and, 178 refractory/relapsed (RR).In accordance with the ERIC recommendations for TP53 disruption [5], mutation analyses were always carried out on samples containing >80% tumor cells; when lower than the 80% cutoff, CD19 positive CLL cells were purified by cell sorting.Briefly, analysis of TP53 mutations was performed with an amplicon-based strategy, covering exons 2-11, as previously reported [4].A minimum coverage of 2,000X was obtained for each sequence in 100% of the analyzed positions, with a limit of detection of 0.3% VAF; TP53 mutated cases with less than 2% VAF were all confirmed by a second independent NGS run starting from DNA [4].Moreover, selected low-VAF TP53 mutations were verified by a different experimental approach (digital droplet PCR, ddPCR).BTK and PLCG2 mutations related to ibrutinib resistance were studied by NGS.Interphase FISH was performed to detect del17p and 11q22.3deletion (del11q) [4].Further methodological details are provided in Supplementary Information.The clinical and biological baseline characteristics of patients [6] are detailed in Supplementry Table S1.All statistical analyses were performed by using standard methods.Overall survival (OS) and progression free survival (PFS) were computed from date of ibrutinib treatment to date of death or progression/ suspension (events), respectively, or last follow-up (censoring).Molecular studies were blinded to the study end points. Among 229 patients, 68 died and 57 progressed after median follow-up of 15.6 months (95% CI 11.9-20.5 months) and 24 months (95% CI 16.0-32.7months) from ibrutinib starting, respectively.As in previous reports [7][8][9], Rai stage, the number of previous treatments (0/1 versus >1), anemia and abnormal LDH values were found to associate with shorter PFS and/or OS by univariable analyses (Table 1 and Supplementry Fig. S1).CLL bearing del17p (n = 74; Supplementry Table S1) showed inferior OS and PFS compared to non-del17p cases (Fig. 1A and Supplementry Table S2), as previously reported [10].Consistently, del17p was independent predictor in multivariable models for OS/ PFS (P = 0.0209, OS; P = 0.0057, PFS; Model 1 Supplementry Table S2).At baseline, before ibrutinib treatment, we identified a total of 296 TP53 mutations in 126 patients (median mutations per patient: 1; range of mutations/patient: 1-11; Supplementry Table S3).The relative high proportion of cases (126/229, 55%) with TP53 mutations can be explained by the use of an ultra-deep NGS strategy allows the detection of very small mutated clone (see also Supplementry Table S4 and Supplementry Fig. S2 for ddPCR validation of selected mutations) [4,5].By classifying TP53-mutated patients according to the VAF of the most prevalent TP53 mutation, VAF range for TP53-mutated cases was 0.53-95.24%(Supplementry Table S3).As in the chemo-immuno therapy setting [4], also in the ibrutinib setting, patients bearing TP53 mutations with low (<10%) and high (≥10%) VAF had shorter OS than TP53wt cases, either kept separate (Fig. 1B), or when low-VAF and high-VAF cases were combined (Supplementry Fig. S3).These results suggest that even low burden TP53 alterations confer a negative impact on outcomes, widening previous findings [11].Accordingly, TP53 mutations were associated with shorter OS/PFS intervals in univariable analyses (Supplementry Table S2), as well as in an OS multivariable model (P = 0.0217; Model 2, Supplementry Table S2).Here, we expanded to low-VAF TP53-mutated patients previous observations on the clinical impact of TP53 disruption upon ibrutinib, as they emerged in the context of clinical trials [7], or in real-life [3,6,8], where TP53 disrupted patients were identified according to the current standard criteria (i.e.VAF ≥ 10%). The combination of del17p with TP53 mutation data identified 95 cases without any TP53 aberrations (non-del17p/non-TP53mut), 8 del17p only cases, 60 TP53-mutated only cases (28 low-VAF), and 66 cases bearing both del17p deletion and TP53 mutations (7 low-VAF).Only patients with concomitant TP53 mutations and del17p showed significantly shorter OS/PFS intervals compared to non-del17p/non-TP53mut cases, while no difference in OS/PFS was found in patients presenting single aberration (Fig. 1C,D).The simultaneous presence of TP53 mutations and del17p confirmed its detrimental clinical impact by univariable analysis and remained independent predictor for short OS/PFS by multivariable analyses together with the number of previous lines of therapy and anemia; consistently, these variables were the most frequently selected by internal bootstrap validation (Table 1).Given the low number of patients of some subgroups (e.g. 8 del17p alone cases), these results need to be confirmed in larger cohorts. At variance from chemo-immunotherapy where the presence of a single TP53 mutation, even with a low-VAF, is associated with a worse outcome [4,12], in the ibrutinib setting only cases presenting a more complex disruption of the TP53 function, due to the concomitant presence of mutations and deletions, fail to have the best benefit from therapy.Our results are in keeping with recent findings suggesting that only double-hit aberrations (i.e. more than one TP53 mutation or TP53 mutation and del17p) are independently associated with a shorter outcome in ibrutinib-treated patients, single-hit aberrations (a single TP53 mutation or del17p only) having an outcome comparable to that of TP53wt patients [2].Differently from Brieghel et al. [2], however, in our cohort, TP53 mutated patients with more than one mutations but without del17p failed to experience a significantly worse prognosis respect to patients without any aberrations (data not shown).In the present series, 52/66 cases concomitantly bearing del17p and TP53 mutations (79%) bore TP53 mutations and/or 17p deletion in most of the neoplastic clone (Supplementry Table S3).We could, therefore, speculate that the genetic instability fostered by such a massive TP53 disruption might eventually lead to the development of more complex genetic lesions, known to correlate with dismal outcomes in the ibrutinib setting [11,13].Our finding may help to explain previous reports of ibrutinib-treated CLL in which TP53 mutations failed to have a prognostic impact [12], and in which the simultaneous presence of TP53 mutations and deletion was not investigated. The evolution of TP53 mutated clones was assessed in 38 patients by longitudinal NGS analysis of paired samples collected at pre-treatment (median time, -0.9 month; range -6.0-0.0) and during (non-relapsed cases; n = 22) or after (relapsed cases; n = 16) ibrutinib treatment (median time interval, 31.8 months, range 3.0-76.9).For relapsed cases, the second time point was collected in close proximity of progression (median time, -0.7 months, range -3.0-1.0 months).No significant differences were observed between relapsed and non-relapsed cases in relation to the timing of the second sampling (P = 0.74).Of a total of 127 TP53 mutations, 92 were present before and 106 after treatment; among these, 21 mutations (median VAF, 1.7%, range 0.4-52.3%)disappeared during the course of treatment, while 35 were newly identified (median VAF, 1.0%, range 0.4-95.2%;Supplementry Table S5).Among relapsed cases, 15/16 showed either a prominent expansion (i.e. a VAF increase greater than 20%) or stability (i.e.VAF variations within the range of 20% VAF variation) over time of the TP53 mutated clone(s) (Fig. 1E).Conversely, in the context of non-relapsed patients, 3 cases presented a VAF increase of the TP53 mutated clones, 13 remained stable, and 6 showed a VAF reduction (Fig. 1F).These data support the idea of a general stability of TP53 subclones under ibrutinib [14], although a positive selection of TP53 mutations over time was slightly overrepresented in relapsed cases (P = 0.04, χ 2 test), suggesting the occurrence of other genetic events complementing the clonal advantage due to TP53 disruption [11,14,15].Considering the 127 TP53 mutations identified across the different time-points, 8 mutations were shared by ≥3 cases (Supplementry Table S5).Among them, G245S and R175H were found expanded (>20% VAF Fig. 1 Clinical impact of TP53 aberrations in ibrutinib-treated CLL.A Kaplan-Meier curves comparing OS probabilities of 155 non-del17p cases (green line), 74 cases with del17p (black line).B Kaplan-Meier curves comparing OS probabilities of 103 TP53wt cases (green line), 91 cases with high-VAF TP53 mutations (TP53mut_highVAF), i.e., ≥10.0% of VAF (black line), and 35 cases with low-VAF TP53 mutations (TP53mut_lowVAF), i.e., <10.0% of VAF (red line).C Kaplan-Meier curves comparing OS probabilities of 95 cases lacking del17p and TP53 mutations (non-del17p/non-TP53mut, green line), 8 cases with del17p only (del17p/non-TP53mut, black line), 60 cases with TP53 mutations only (non-del17p/TP53mut, red line), and 66 cases with concomitant TP53 mutation and del17p (del17p/TP53mut, blue line).D Kaplan-Meier curves comparing PFS probabilities of 95 cases lacking del17p and TP53 mutations (non-del17p/non-TP53mut, green line), 8 cases with del17p only (del17p/non-TP53mut, black line), 60 cases with TP53 mutations only (non-del17p/TP53mut, red line), and 66 cases with a concomitant TP53 mutation and del17p (del17p/TP53mut, blue line).In Kaplan-Meier curves, cases with more than one mutation are classified according to the mutation with the highest VAF (see Supplementry Table S3).The number of patients in each group is reported; P values refer to the logrank test.E Clonal evolution of TP53 mutations in longitudinal samples relapsed under ibrutinib treatment (relapsed cases).Graph reports results for 16 CLL patients (65 TP53 mutations) longitudinally studied at two different time-points, the 1 st (x-axis) collected at ibrutinib start and the 2 nd (y-axis) collected after ibrutinib interruption for relapse; overall, VAF values are referred to 65 TP53 mutations, as measured at the two time-points.F Clonal evolution of TP53 mutations in longitudinal samples during ibrutinib treatment (non-relapsed cases).Graph reports results for 22 CLL patients (62 TP53 mutations) longitudinally studied at two different time-points, the 1 st (x-axis) collected at ibrutinib start and the 2 nd (y-axis) collected during ibrutinib treatment; overall, VAF values are referred to 62 TP53 mutations, as measured at the two timepoints.The red color denotes a mutation with a VAF increase greater than 20%.The green color denotes a mutation with a VAF decrease greater than 20%.The dotted parallel lines denote the 20% interval on either side.increase) in 3/4 and 2/3 cases, respectively (Supplementry Table S5), suggesting their possible role in ibrutinib resistance.BTK and PLCG2 mutations, were retrieved in 3/7 relapsed cases presenting a positive selection for TP53 mutations at the relapse time (Supplementry S5).Overall, BTK and PLCG2 mutations were discovered in 9/16 (56%) relapsed cases versus 3/22 (14%) patients under ibrutinib treatment (P = 0.006, χ 2 test; Supplementry Table S5). In conclusion, here we provided evidence that only the copresence of TP53 deletion and mutations, the latter even with a low-VAF representation, and not the single aberrations have a negative prognostic impact in CLL patients under ibrutinib treatment.In practice, this finding points toward the need of a complete assessment of TP53 aberrations to be performed in all CLL patients prior to start ibrutinib treatment.A lower threshold for reporting TP53 mutations (e.g.VAF < 10%) must be evaluated in prospective clinical trial cohorts before it can be accepted as standard for routine practice.Moreover, low-VAF TP53 mutations should be always confirmed by orthogonal assays (e.g.ddPCR) or by repetition [4]. ", "section_name": "", "section_num": "" } ]	[ { "section_content": "The data that support the findings of this study are available from the corresponding author upon request. ", "section_name": "DATA AVAILABILITY", "section_num": null }, { "section_content": "RB, designed the study, interpreted data, and wrote the manuscript; FMR, FV, TB, AZ, RP, ET, FP, MD, GF, performed and interpreted molecular studies, and contributed to data interpretation; FV, JP, and RB generated the bioinformatics pipeline of analysis, and performed statistical analyses; PB, RM, GR, LL, JO, AC, RL, MP, MIDP, AC, MG, FM, AT, FZ, RF, FDR, GDP collected clinical data and contributed to data interpretation; VG designed the study, interpreted data, and wrote the manuscript.All the Authors agreed on the final form of the manuscript with the only exclusion of GDP (deceased). The present study is supported in part by: Progetto Ricerca Finalizzata The authors declare no competing interests. The online version contains supplementary material available at https://doi.org/10.1038/s41375-023-01845-9. Correspondence and requests for materials should be addressed to Riccardo Bomben or Valter Gattei. Reprints and permission information is available at http://www.nature.com/reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "RB, designed the study, interpreted data, and wrote the manuscript; FMR, FV, TB, AZ, RP, ET, FP, MD, GF, performed and interpreted molecular studies, and contributed to data interpretation; FV, JP, and RB generated the bioinformatics pipeline of analysis, and performed statistical analyses; PB, RM, GR, LL, JO, AC, RL, MP, MIDP, AC, MG, FM, AT, FZ, RF, FDR, GDP collected clinical data and contributed to data interpretation; VG designed the study, interpreted data, and wrote the manuscript.All the Authors agreed on the final form of the manuscript with the only exclusion of GDP (deceased). ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "The present study is supported in part by: Progetto Ricerca Finalizzata ", "section_name": "FUNDING", "section_num": null }, { "section_content": "The authors declare no competing interests. ", "section_name": "COMPETING INTERESTS", "section_num": null }, { "section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41375-023-01845-9. Correspondence and requests for materials should be addressed to Riccardo Bomben or Valter Gattei. Reprints and permission information is available at http://www.nature.com/reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "ADDITIONAL INFORMATION Supplementary information", "section_num": null } ]
10.1371/journal.pone.0258423	Detailing the epidemiological and clinical characteristics of chronic lymphocytic leukaemia in Portugal—Results from a population-based cancer registry cohort study	<jats:sec id="sec001"> <jats:title>Background</jats:title> <jats:p>Chronic lymphocytic leukaemia (CLL) is the most common leukaemia among adults in western countries. Considering the increasing incidence and prevalence of this condition, it is highly relevant to better characterise these patients in Portugal, where data is still scarce.</jats:p> </jats:sec> <jats:sec id="sec002"> <jats:title>Methods</jats:title> <jats:p>To determine incidence, clinical presentation, survival and second malignancies, a population-based historical cohort study was conducted. Cases of interest were identified through the South Region Cancer Registry database and additional data sources. Patients aged ≥18 years, with a confirmed diagnosis of CLL or small lymphocytic lymphoma between January 1<jats:sup>st</jats:sup>, 2013 and December 31<jats:sup>st</jats:sup>, 2014 were included. Patients were followed‐up until death or cut-off date (December 31<jats:sup>st</jats:sup>, 2019).</jats:p> </jats:sec> <jats:sec id="sec003"> <jats:title>Results</jats:title> <jats:p>A total of 496 patients were included and median follow-up time was 5.46 years. Crude incidence rates were 5.03 and 5.22 per 100,000 inhabitants for 2013 and 2014, respectively, and age-adjusted incidence rates were 3.18:100,000 European population for 2013 and 3.35:100,000 European population for 2014. Median age at diagnosis was 71 years and the male/female ratio was 1.40. The majority of patients had leukemic presentation of the disease (86.09%), was diagnosed in Binet stage A (75.58%) and did not present B symptoms (84.01%), anaemia (haemoglobin ≤10g/dL; 90.63%) nor thrombocytopenia (platelet count ≤100 000/μL; 91.73%). Five-year overall survival (OS) rate was 70.53% (95%CI 66.31–74.34) and age, lactate dehydrogenase, Binet stage and a ≥5 Charlson comorbidity index score were independently associated with OS. Standardised-incidence ratios for any second malignancy and cutaneous squamous cell carcinoma were 1.59 (95%CI 1.19–2.08) and 10.15 (95%CI 6.28–15.51), respectively.</jats:p> </jats:sec> <jats:sec id="sec004"> <jats:title>Conclusion</jats:title> <jats:p>Incidence, clinical presentation and survival of CLL Portuguese patients are similar to those reported for other western countries. The increased risk of second malignancies raises concerns and needs adequate clinical watchfulness.</jats:p> </jats:sec>	[ { "section_content": "Chronic lymphocytic leukaemia (CLL) is an indolent lymphoproliferative malignancy of CD5+ mature B cells involving peripheral blood, bone marrow and lymphoid organs.It is the most common leukaemia among adults in western countries, with incidence rates ranging between 4 and 6 per 100,000 people per year; in contrast, it is rare in Asia, particularly in Japan and Korea.Incidence increases with age, to more than 30:100,000 inhabitants per year at an age of >80 and, at diagnosis, at least 70% of patients are older than 65 years [1][2][3][4]. The diagnosis requires a minimum of 5000 B lymphocytes/μL in the peripheral blood and its clonality needs to be confirmed by flow cytometry [5][6][7].Additionally, small lymphocytic lymphoma (SLL) represents a different clinical presentation of the same disease and follows the same management guidelines [3].This entity is characterised by the presence of lymphadenopathy and/or organomegaly with less than 5000 B lymphocytes/μL in the peripheral blood with a CLL phenotype [5][6][7]. CLL course is extremely heterogeneous.At diagnosis, the majority of patients are asymptomatic and the disease is detected in a routine blood count.In advanced stages, patients may present anaemia and thrombocytopenia and, scarcely ever, B symptoms.To better stratify patient's death risk, numerous clinical and biological prognostic predictors have been identified, including Rai and Binet staging systems [8,9], lactate dehydrogenase and β2-microglobulin serum levels [10], tumour genomic aberrations [del(11q), del(13q), del(17p), 12 trisomy)] [11], the mutational status of the immunoglobulin heavy-chain variable region genes (IGHV) [12,13], protein expression (CD38, ZAP-70) [12,14], and the mutational status of the TP53 tumour-suppressor gene [15].The CLL international prognostic index (CLL-IPI) includes some of these variables and has been validated as predictive of clinical outcomes in various settings in the era of immunochemmotherapy [16]. As other indolent lymphoid disorders, CLL has favourable survival results, with a 5-year relative survival of 80-86% and a death rate of 1.1 per 100,000 people per year [4,17], despite the fact that these patients have an increased risk of developing second malignancies [18,19]. The increasing incidence and prevalence of CLL has augmented the impact of this condition globally.Therefore, it is exceptionally relevant to better understand epidemiology and patients' characteristics in order to adequate health, social and public health interventions, particularly in Portugal, where this data is still scarce.The aim of this study was to determine the incidence, clinical presentation at diagnosis, survival and occurrence of second malignancies in CLL recurring to a diagnosis cohort from the south region of Portugal. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Material and methods", "section_num": null }, { "section_content": "A population-based historical cohort study was conducted and is reported in accordance with STROBE statement [20]. ", "section_name": "Study design", "section_num": null }, { "section_content": "The South Region Cancer Registry (ROR-Sul), established in Portugal in 1988, is a populationbased cancer registry covering 4.8 million inhabitants from the regions of Lisboa e Vale do Tejo, Alentejo, Algarve and autonomous region of Madeira.ROR-Sul's database complies with international recommendations stated by the International Agency for Research on Cancer [21] and collects relevant information from time of cancer diagnosis until death.Cancer cases are signalised through institution-based databases (mainly from pathology departments) and numerous variables are semi-automatically transferred into the database (e.g.date of birth, sex, place of residence, topography, morphology, vital status and date of death).Other relevant information, mainly data related to disease stage, prognostic features and treatments are registered manually by trained and experienced personnel at each health institution.The current study was conducted recurring to ROR-Sul's database. This study was approved by the Instituto Português de Oncologia de Lisboa Francisco Gentil Ethics Committee on September 6th, 2018 (reference: UIC/1201).Informed consent was not required because this is a historical cohort study, all variables used in this study are already part of the ROR-SUL database and are specifically collected to accomplish registry purposes, as dictated by National law. ", "section_name": "Data sources and ethics", "section_num": null }, { "section_content": "Patients aged 18 years or older diagnosed with CLL or SLL (morphology codes: M9670/3 and M9823/3) [22] between January 1st, 2013 and December 31st, 2014 were included.Cases without histological or immunophenotypical confirmation were excluded.Cases exhaustiveness was maximized per two independent strategies: (a) cases registered in ROR-Sul database with a diagnosis in the study period of non-otherwise specified lymphomas and leukaemias were centrally reviewed and cases of interest were recoded (b) flow cytometry laboratories from public and private hospitals provided results from immunophenotypic analysis conducted during the study period and additional cases of interest were registered in ROR-Sul's database.Patients were followed-up between diagnosis and death or cut-off date (December 31 st , 2019). Information of interest included: age, sex, date of diagnosis, morphology, comorbidities (according to the Charlson index), leukocyte, lymphocyte and platelet counts, haemoglobin, B symptoms, Eastern Cooperative Oncology Group Performance Status (ECOG PS), lactate dehydrogenase, β2-microglobulin, Rai stage, Binet stage, del(17p), del(11q), del(13q), trisomy 12, TP53 mutations, IGHV mutational status, treatment realisation and type of first line treatment, topography and morphology of second malignancies, vital status and date of death/last known contact.Data for variables of interest was centrally validated by the research team. ", "section_name": "Study population", "section_num": null }, { "section_content": "Incidence was determined recurring to crude and age-adjusted (European population) rates.Demographic and clinical characterization was conducted considering information available at diagnosis.Morphology was coded considering a lymphocyte cut-off of 5000x10 6 /L, according to WHO classification [5][6][7] Comorbidities were collected considering those included in the Charlson comorbidity index (CCI) [23].Characterisation of chromosomal aberrations per fluorescence in situ hybridization (FISH) and mutational status of the IGHV and TP53 were conducted considering the evaluations performed before the initiation of first treatment. Overall survival was defined as the time elapsed between diagnosis and death due to any cause.The second malignancies considered were those occurring at least 6 months after CLL/ SLL diagnosis (in order to exclude synchronous neoplasms) [24] and included all malignant tumours except basal cell carcinomas. ", "section_name": "Study outcomes", "section_num": null }, { "section_content": "Data was analysed in a pseudonymized format.Crude incidence rates were computed for the years of 2013 and 2014 using the direct method and considering the resident population in ROR-Sul coverage area publicly provided by Statistics Portugal (INE) [25].Additionally, agestandardised incidence rates were computed for the European standard population [26]. Demographic and clinical characteristics and second malignancies were summarised using absolute and relative frequencies for categorical variables and median and interquartile ranges (IQR) for continuous variables.We established a cut-off of 20% for missing data and, when the unknown information was lower than the cut-off, it was presented as a complete-case analysis.Otherwise, it was computed for the study population [27].CCI was calculated according to the original method and summarised with frequencies, mean and standard deviation.Posthoc bivariate analysis relating Binet stage with sex and age (<65 and �65 years) were performed using chi-square test. Kaplan-Meier estimates were used to compute OS and patients without the event of interest were censored at cut-off date.5-year survival rates were calculated considering a 95% confidence interval (CI).The log-rank test was used to compare differences between strata.Posthoc subgroup analyses for OS according to age (<65 and �65 years) were performed. A multivariable proportional hazard regression was used to evaluate the association between variables of interest and OS.Covariates with more than 30% of unknown data were not considered for modeling.Variables included in the multivariable regression were the significant covariates in univariate analysis (cut-off p<0.20) or clinically relevant based on their known prognostic value.Assumptions of the model were verified. Standardised incidence ratio (SIR) was calculated as the ratio between observed and expected number of second primary malignancies.The expected number of cancers was computed by multiplying ROR-Sul crude incidence rates for 2013 stratified by sex and age groups by stratum-specific person-years at risk of the CLL cohort and summing across strata.For each cancer type, SIR was not calculated to those with less or equal to 5 observed cases.95%CI was calculated assuming a Poisson distribution for the observed cases. All statistical analyses were performed using the software Stata version 13.0 [28]. ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "We first identified in ROR-Sul database 401 cases that met the inclusion criteria.Posteriorly, 126 additional cases were identified through the flow cytometry laboratories' results and, lastly, 31 cases were excluded as they met the exclusion criteria.The final number of patients included in the study was 496 (Fig 1).Median follow-up of the study population was 5.46 years; only 2 patients (0.40%) were lost to follow-up. ", "section_name": "Results", "section_num": null }, { "section_content": "Crude incidence rates were determined to be 5.03 and 5.22 per 100,000 inhabitants for 2013 and 2014, respectively.Age-adjusted incidence rates (European population), were 3.18:100,000 people for 2013 and 3.35:100,000 people for 2014.Clinical presentation at diagnosis is shown in Table 1.Median age at diagnosis was 71 years, with 70.77% of patients older than 65 years, and the sex distribution (male/female ratio) was 1.40.The majority of patients had a leukemic picture (80.04%) and were diagnosed in Binet stage A (75.85%).Male sex was associated with more advanced Binet stages (p = 0.044); no statistical significant relationships were found between age and stage (p = 0.290). At least one chromosomal aberration was evaluated by FISH in 203 patients (40.93%) and the four abnormalities of interest were studied in 198 patients (39.92%).Of these, 62.12% exhibited abnormalities and the most prevalent alteration was 13q deletion (Table 2).In 104 patients there was one aberration, 12 patients had two and 3 patients had three aberrations. The mutational status of the IGHV and TP53 were evaluated in 82 (16.53%) and 39 (7.86%) patients, respectively.With respect to IGHV, 43 patients (52.44%) were hypermutated and 39 (47.56%) were non-hypermutated.TP53 mutation was present in 5.13% of evaluated patients. ", "section_name": "Incidence and clinical presentation", "section_num": null }, { "section_content": "During the study period, the majority of patients did not receive any treatment (mostly due to a watch and wait strategy) and 188 patients (37.90%) received at least one therapeutic line for CLL.The most common first-line treatment options were chemmoimmunotherapy (46.28%), including fludarabine, cyclophosphamide, rituximab (n = 49; 26.06%) and bendamustine, rituximab (n = 14; 7.45%), alkylating agent +/-corticosteroids (28.19%) and anthracycline or vinca alkaloids-containing regimens (17.02%). OS estimate is presented in Fig 2 .Median OS was not reached and 5-year survival rate was 70.53% (IC95% 66.31-74.34). OS estimate was stratified according to Binet and Rai staging systems (S1 and S2 Figs).Median OS was not reached for the majority of strata although it was reached for high-risk/ advanced stages [Binet C: 1.62 years (0.63-not reached); Rai III/IV: 1.62 years (0.64-not reached)].Additionally, OS was stratified according to age (<65 and �65) and, although medians were not reached, the 5-year OS rates were 86.66% (95%CI 83.43-93.63)and 62.62% (95%CI 57.33-67.45)for younger and older patients, respectively. Univariate and multivariable analysis evaluating the association between baseline characteristics and OS is presented in Table 3. Due to the high number of cases with missing information, β2-microglobulin levels were not included in this analysis.Age, lactate dehydrogenase, Binet stage and a CCI �5 score were identified as independent factors associated with OS (p<0.001,p = 0.004, p = 0.002 and p = 0.021, respectively). ", "section_name": "Treatment and survival", "section_num": null }, { "section_content": "A total of 53 second malignant tumours were identified in 46 patients, which corresponds to a cumulative incidence of 10.69%.The most common second malignancies were cutaneous squamous cell carcinoma (CSCC) (n = 21), prostate cancer (n = 5) and malignant tumours of the central nervous system (n = 4) (S1 Table ).The risk of any second primary cancer in CLL patients was increased comparing to the general population [SIR = 1.59 (95%CI 1.19-2.08)].Site-specific SIR was calculated to CSCC and estimated to be 10.15 (95%CI 6.28-15.51). ", "section_name": "Second malignancies", "section_num": null }, { "section_content": "Cancer registries are important tools in the generation of epidemiological data.To the best of our knowledge, this is the first population-based study that aimed to characterise the incidence, clinical presentation, survival and the occurrence of second malignancies in CLL patients in Portugal.As this condition is particularly incident and prevalent in western countries, with a significant impact on both social and health systems, it is of utmost relevance to better understand its epidemiology and patients' characteristics.With respect to cases completeness, it is worth noticing that, in this study, patients identified through flow cytometry laboratories represent over 25% of the total cases; in fact, a 20-30% under-reporting of CLL cases for other population-based cancer registries was already described [29][30][31].Thus, the use of additional information sources as a routine strategy is mandatory in order to provide accurate incidence and survival data for CLL/SLL.Nevertheless, it should be highlighted that the present work contributed to an accurate CLL/ SLL incidence and survival determination in Portugal and illustrates the importance of adapting CLL/SLL epidemiologic surveillance to provide reliable and comparable data.Incidence rates were comparable to other western countries, including the United States of America (USA), Canada and European countries such as Spain and Sweden, but contrasted with the incidence in the United Kingdom and Belgium (estimated at 6:100,000 European population) [29,[32][33][34][35][36][37], which may be related to differences in characteristics of the population and/or risk factors.Differences were also observed when comparing with data from Eastern Europe, Russia and Asian countries, where it a lower incidence of this malignancy has been described.Age at diagnosis and sex ratio were also comparable to other western countries [32,35,37]. The general outcomes of this population, predominantly of early stage CLL and in patients with good performance status, were very favourable.Median OS was not reached, as could be expected due to the relatively short follow-up duration in an indolent malignancy (5.46 years).Nevertheless, the 5-year OS rate is concordant with survival results reported for other developed countries (mainly western countries such as Germany, Sweden, Norway) and contrasts with those from developing countries [17,34,[37][38][39]. The analysis of the clinical characteristics at diagnosis showed similarities to data reported for other developed countries [40][41][42] and diverges from data from developing countries, in which patients are diagnosed in more advanced/high risk stages, with higher prevalence of anaemia and thrombocytopenia [38,42,43].The proportion of patients with unknown information for β2-microglobulin (56.05%) should be noted; although it is independently associated with OS and response duration [44], it was not systematically evaluated in the study population.It is also imperative to mention that the majority of patients (53.43%) did not present any of the comorbidities of the CCI and had ECOG PS�2.Even though other authors reported the absence of comorbidities at diagnosis in 70% of CLL patients [45], we recognise that the application of CCI in CLL is limited.The Cumulative Illness Rating Scale (CIRS), not available in ROR-Sul database, is more frequently used [46].Another issue that might have contributed to these results is the retrospective collection of this information, which could have led to information bias. Chromosomal aberrations were evaluated by FISH in 40.93% of patients before treatment, which represents a limitation for data interpretation.Nonetheless, this proportion of evaluated patients was expected as, during the study period, the recommendations to conduct this characterisation were still limited and had no impact in therapeutic decisions in our country [47].Generally, the prevalence of these alterations is in agreement with studies performed in other populations, although it should be noted the higher proportion of patients without FISH abnormalities (37.69% vs 15-25%) [17,18,39,43,45].As mentioned before, this analysis and interpretation of the results have limitations and, to achieve robust conclusions, more studies should be performed, ideally prospective, with larger series of patients. Treatment characterisation showed that a watch and wait strategy was chosen for the majority of the cases, as recommended in current guidelines and as it would be expected considering the follow-up duration (5.46 years).First-line treatment options generally agreed with international recommendations for that time period, although regimens containing anthracyclines and vinca alkaloids were also unexpectedly used and should be explored in additional/ future research. OS stratification per Rai and Binet staging systems, as well as the multivariable analysis, apparently validate its use in this population (p<0.001 and p = 0.004, respectively).Median OS was reached for Binet stage C [1.62 years (95%CI 0.63 -not reached)] and Rai stage III/IV [1.62 years (95%CI 0.64 -not reached)] strata.Although these results differ from others reported in the literature (e.g.Binet C having a median OS of 7.9 years [17]), the interpretation of this data should be cautious due the low number of patients in these stratum (n = 41) and the short follow-up duration, which contributed to a less precise estimate, as can be appreciated looking at the CI amplitude.Additionally, treatment options, that may have had an important impact on survival outcomes, were only restrictedly analysed in this series. Our study confirmed age, lactate dehydrogenase and Binet stage as being independently associated with OS.In our analysis, a CCI score �5 was independently associated with OS although CCI 1-4 strata were not.This result is in agreement with those reported in the literature per other authors [48][49][50]. Regarding second malignancies, the proportional incidence of CSCC, malignant tumours of the central nervous system, myelodysplastic syndromes and malignant melanomas of the skin is superior to that reported to the general population.The SIR observed to any second cancer identified a 1.59 fold increased risk and this result is concordant with data from other series [51][52][53].Site-specific SIR calculated for CSCC revealed a 10.15 increased risk for the development of this tumour.There are few studies in the literature that evaluated the risk of developing CSCC after CLL.Zheng et al. observed a 7.63 fold increased risk (95%CI 7.06-8.25)for the Swedish population and Ishdorj and colleagues found a five-fold increased risk in the Canadian population [54,55], inferior to our results although with coincident CI.It should be noted that our observations arise from population-based data, which contributed to cases exhaustiveness of both CLL and second malignancies and consequently to the accuracy of the estimates.However, we admit that the number of patients included in this study may have contributed to a less precise estimate as shown by the CI amplitude.Nevertheless, these results raise concerns and highlight the importance of adequate clinical watchfulness during the follow-up of CLL patients. This study has various strengths: it was conducted at the population level, included patients from both private and public hospital institutions and cases of interest were signalised through several data sources, which constitute major contributions to the external validity of our results.It is worth noting the central procedures for data accuracy and validation, which contributed to the internal validity of our work.It is also important to highlight the proportion of follow-up completeness (99.6%), extremely relevant to characterise the outcomes of interest.Nonetheless, there are also some limitations, namely the proportion of unknown/not evaluated information for some of the variables of interest and the absence of some other relevant data (e.g.autoimmune phenomena), which is inherent to observational/registry studies, but can lead to information bias.Additionally, it is worth mentioning the possible misclassification bias of some of the variables of interest used to analyse the outcomes.However, procedures were implemented to its minimisation, namely the central validation of the information for those variables.Another limitation concerns the relatively short follow-up considering the characteristically indolent evolution of this disease; hence, a longer follow-up would be important to achieve more robust results. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Our study confirms that epidemiological and clinical characteristics of Portuguese CLL patients are concordant to those reported from other western countries.Clinical outcomes, measured by 5-year overall survival rates, are also similar to other western countries.The increased risk of second malignancies raises concerns and needs adequate clinical watchfulness. ", "section_name": "Conclusions", "section_num": null }, { "section_content": "", "section_name": "Supporting information", "section_num": null } ]	[ { "section_content": "The authors thank all health institutions and immunophenotypic laboratories of the ROR-Sul coverage area for all their contribution and commitment to this research project. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "Abbvie, Lda (https://www.abbvie.pt/)provided funding to support this report.The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "Data cannot be shared publicly due to legal and ethical issues.Data are available from the National Cancer Registry Institutional Data Access (contact via [email protected]) for researchers who meet the criteria for access to confidential data. ", "section_name": "", "section_num": "" } ]
10.3390/hematolrep14010006	Challenges in Diagnosis and Clinical Management of COVID-19 in Patient with B-Cell Chronic Lymphocytic Leukemia (CLL): Report of One Case	<jats:p>We report here a case of a patient affected by B-cell chronic lymphocytic leukemia (CLL) that developed COVID-19 during the actual SARS-CoV-2 outbreak. The coexistence of CLL and COVID-19 raises many questions regarding the possible increased risk of developing COVID-19 among patients with CLL, the problems in managing therapies for both diseases and, above all, the difficulties in diagnosing COVID-19 in patients affected by CLL. In our patient, an 84-year-old man, the recognition of COVID-19 was delayed because of its atypical clinical presentation and technical problems related to the methods used for the diagnosis. Based on the symptoms and the radiological aspect of the lung, the occurrence of COVID-19 was suspected. Repeated tests on oro/nasopharyngeal swabs gave negative results, causing a delay in the diagnosis. Moreover, different methods used to identify the SARS-CoV-2 antibodies in serum gave conflicting results, and only two tests were able to identify SARS-CoV-2 Abs of the IgG type. During the clinical course of unrecognized COVID-19, our patient developed severe complications and did not receive any specific treatment for the two diseases. Recognition of COVID-19 in patients with CLL is a challenging task and the most accurate methods are necessary to overcome the diagnostic difficulties encountered.</jats:p>	[ { "section_content": "An 84-year-old obese man was diagnosed with CLL in 2006.In 2017, he underwent transcatheter aortic valve implantation (TAVI) because of the worsening of his atrial fibrillation due to aortic stenosis.Bone marrow aspiration, flow cytometry immunophenotyping and hematological investigations were performed over the course of disease, including a Binet stage C, Rai stage IV and a European Cooperative Oncology Group (ECOG) performance score of 1.The CLL was treated for several years only with prednisone at 10 mg per day. In February 2020, based on a peak of lymphocytosis and flow cytometry analysis, treatment with ibrutinib was considered.However, he never started such treatment because in March 2020, he became symptomatic with a sore throat, fever (39 • C), dyspnea and a dry cough, associated with a paO2 < 80%.For this reason, he was hospitalized in the intensive care unit.On admission, he showed acute hypoxemic respiratory failure, with a pO2 = 35, and started non-invasive ventilation (NIV) therapy 12-10, with FiO2 55%. High-resolution CT (HRCT) of the lung showed the typical aspect of interstitial pneumonia, with a \"ground glass\" appearance.The total severity score (TSS) and the COVID RADS were calculated in January 2020, before the onset of the symptoms, and in March 2020 during the acute phase of the disease according to the methods previously described [1,2].TSS results were 0 and 16, and RADS scores were 0 and 4, respectively.The HRCT scan also showed signs of distal, bilateral lung thromboembolism (Figure 1), and treatment with fondaparinux (Arixtra) at 1.5 mg/day was administered.Based on the results of the HRCT scan and thromboembolic complications, considering the TSS and CO-RADS scores, the occurrence of SARS-CoV-2 infection was suspected, and nasal/oropharyngeal swabs followed by a real-time reverse-transcription polymerase chain reaction (rRT-PCR) test were performed.We used two different kits, namely the Allplex™ 2019-nCoV Assay (Seegene MuDT TM , Seoul, Republic of Korea) and the RNA Detection kit (DAAN Gene Co. LTD, Guangzhou, Guangdong, China).The result was negative with both.In agreement with WHO recommendations in case of suspected cases, we repeated the test many times, with the same negative results (Table 1).Therefore, the patient was not transferred to the isolation ward for appropriate patient management in accordance with the specific COVID-19 Integrated Care Pathway (ICP), recently published [3].He was admitted first to intensive care and then the pneumatological unit, where a bronchoalveolar lavage (BAL) was performed without any special protection.Physicians attending to him were therefore potentially exposed to infection.The BAL specimen was subjected to cytological assessment and culture, in search of other possible bacterial and fungal infections.On that occasion, another attempt was made to detect the SARS-CoV-2 virus in the BAL specimen using the kit by Seegene.The rRT-PCR failed to identify the virus, probably because of the delayed timing of the exam. During the acute phase of the disease, our patient did not show the typical laboratory findings of a COVID-19 patient, consisting of marked lymphocytopenia and a marked reduction in both CD8+ and CD4+ T cells.The laboratory test, on the contrary, showed marked lymphocytosis and thrombocytopenia, reported in Table 1.The lymphocytosis observed in our patient with CLL was responsible for the masking of the occurrence of COVID-19 [4,5].Nevertheless, during hospitalization, the clinical presentation and the radiological aspect of the lung strongly suggested the presence of SARS-CoV-2 infection.We therefore requested the identification of specific immunoglobulins in serum directed against the SARS-CoV-2 virus using different kits (EUROIMMUN Anti-SARS-CoV-2 Ig ELISA; Abbott SARS-CoV-2 Ig assay; Maglumi™ 2019-n-Cov: IgG and IgM automated quantitative chemiluminescent immunoassays (CLIA); Human Anti-2019 nCoV(S) Ig by ELISA Fine Test-Wuhan Fine Biotech Co., Ltd., Wuhan, China).IgM of SARS-CoV-2 always resulted negative, whereas a repeated search of IgG using the different kits gave contradictory results.Only two assays (Maglumi and Fine Test) were able to identify the presence of SARS-CoV-2 IgG antibodies.Therefore, in addition to the difficulties in identifying the disease based on the laboratory results, we had to face the difficulties related to the specificity and sensitivity of the different tests recommended for the diagnosis of COVID-19. In our patient, the clinical course of COVID-19 was severe.While at the intensive care unit, he received non-invasive ventilatory (NIV) therapy until the dyspnea subsided.He also developed a thromboembolic complication and an Acinetobacter baumanii infection of the lung.He did not receive any treatment for COVID-19, and he could not be treated with ibrutinib for CLL as well.The patient is now in good clinical condition, with some respiratory complications but a globally well-preserved pulmonary function. Table 1.The diagnostic procedures (RT-PCR, SARS-CoV-2 IgG, blood cell counts, symptoms) and medical history are summarized.During the acute phase of the disease, our patient did not show the typical laboratory findings of a COVID-19 patient, consisting of marked lymphocytopenia and a marked reduction in both CD8+ and CD4+ T cells.The laboratory test, on the contrary, showed marked lymphocytosis and thrombocytopenia, reported in Table 1.The lymphocytosis observed in our patient with CLL was responsible for the masking of the occurrence of COVID-19 [4,5].Nevertheless, during hospitalization, the clinical presentation and the radiological aspect of the lung strongly suggested the presence of SARS-CoV-2 infection.We therefore requested the identification of specific immunoglobulins in serum directed against the SARS-CoV-2 virus using different kits (EUROIMMUN Anti-SARS-CoV-2 Ig ELISA; Abbott SARS-CoV-2 Ig assay; Maglumi™ 2019-n-Cov: IgG and IgM automated quantitative chemiluminescent immunoassays (CLIA); Human Anti-2019 nCoV(S) Ig by ELISA Fine Test-Wuhan Fine Biotech Co).IgM of SARS-CoV-2 always resulted negative, whereas a repeated search of IgG using the different kits gave contradictory results.Only two assays (Maglumi and Fine Test) were able to identify the presence of SARS-CoV-2 IgG antibodies.Therefore, in addition to the difficulties in identifying the disease based on the laboratory results, we had to face the difficulties related to the specificity and sensitivity of the different tests recommended for the diagnosis of COVID-19. In our patient, the clinical course of COVID-19 was severe.While at the intensive care unit, he received non-invasive ventilatory (NIV) therapy until the dyspnea subsided.He also developed a thromboembolic complication and an Acinetobacter Baumanii infection of the lung.He did not receive any treatment for COVID-19, and he could not be treated with ibrutinib for CLL as well.The patient is now in good clinical condition, with some respiratory complications but a globally well-preserved pulmonary function. ", "section_name": "Case Report", "section_num": "1." }, { "section_content": "Written informed consent was obtained from the participants of the study.The study was approved by our Institutional Ethical Committee (University Sapienza of Rome, Italy) (Prot.#52SA_2020, RIF.CE5773_2020), on the basis that it complied with the Declaration of Helsinki and that the protocol followed existing good clinical practice guidelines. ", "section_name": "Ethical Approval", "section_num": "2." }, { "section_content": "The clinical management of this patient raised several points that deserve discussion.The first point concerns the diagnosis of COVID-19 and the accuracy of the currently available tests.It appears that the detection of SARS-CoV-2 in nasal/oropharyngeal swabs, considered the reference standard for COVID-19 diagnostics, is not so accurate and sensitive in all cases.Repeated tests in our patient constantly gave negative results.In other similar cases of coexisting CLL and COVID-19, real-time RT-PCR was able to detect the SARS-CoV-2 virus [4].This was not the case in our patient.According to WHO guidelines (Laboratory testing for coronavirus disease (COVID-19) in suspected human cases, 2020) [6] and based on the protocol developed by Charitè, Berlin, Germany [7], the diagnosis should be based on the demonstration of the virus in nasal/oropharyngeal swabs, analyzed by rRT-PCR.In severe cases, the most suitable samples, showing a higher RNA-positive rate, seem to be BAL or deep sputum [8].The two most frequently used and recommended kits, both CE-approved, are the one by Seegene (Allplex™ 2019-nCoV Assay) and the one by DAAN (RNA Detection kit).The first one is based on the identification of at least two out of the three genes considered (E, RdRP and N genes), while the second one is based on the identification of only one of the two genes examined (ORF1ab and N genes).In our patient, both kits repeatedly failed to demonstrate the presence of the virus in the nasal/oropharyngeal swabs and the BAL too.Therefore, the initial diagnosis was delayed because of the inability of these two kits to detect the virus.It has been previously reported that molecular tests carried out through swabs can give false negative results in people who harbor the virus.This happens because swab collection and time of sampling are critical.This could be a possible explanation of the false negative results at least in the last test, which was performed too late (weeks later) when the virus could no longer be present in the BAL specimen.Although such repeated negative results may be related to this specific patient, they could reflect a lack of sensitivity in the panels used for RT-PCR [9,10].However, as suggested by Niu A et al. [9], since patients with hematological diseases have a high risk of developing a severe form of COVID-19 and are also more susceptible to false negative RT-PCR tests, the diagnostic and therapeutic management of this selected population should be more aggressively performed. We were able to make the diagnosis only after the acute phase, when there was a rise in the specific IgG.The presence of specific IgG directed against the viral antigens was demonstrated, in fact, only after discharge from the hospital.Serology tests are useful in identifying individuals who may have developed an immune response to SARS-CoV-2.Furthermore, it has been suggested that they may be of great help to overcome lockdowns [11] and to allow a population-wide estimation of infected people.However, they are not considered useful for diagnosing COVID-19, mostly because they cannot be detected in the early days of infection.They may be useful if the diagnosis is missed at admission and can only be obtained later during recovery. The second point concerns the accuracy of the different commercially available methods to detect the immunoglobulins.The sensitivity of the methods used to detect specific immunoglobulins in serum, in fact, varies according to the method used.The method that, in our experience, was able to detect specific IgG in the serum was the one that is directed toward two different antigens, namely the nucleocapsid (N) protein and the spike (S) protein of the virus.The other methods we used, based on the detection of specific IgG directed toward only the N protein, gave a negative result.This is in agreement with previous observations regarding the accuracy of these methods [12,13].In many countries, the use of these tests in the general population has been planned to obtain epidemiological information regarding the diffusion of the virus in order to identify and isolate persons who are contagious and to establish the real lethality rate of the disease.We should consider that the World Health Organization (WHO) recently stated that there is not enough evidence about the effectiveness of antibody-mediated immunity to guarantee the accuracy of an 'immunity passport' or 'risk-free certificate' (World Health Organization.\"Immunity passports\" in the context of .In any case, considering the potential pitfalls and benefits of such tests as well as the ethical and economic issues [15], the use of serological antibody testing is rapidly emerging [16]. In Italy, an epidemiological study is currently ongoing for the detection of anti-SARS-CoV-2 N antigen-specific IgG in a representative group of 150,000 people [17].It would be extremely relevant if such information could be based on the most accurate test available.An exhaustive list of all the kits has recently been published by the FDA [18].They can be divided into those that recognize only one single target (the nucleocapsid, the spike protein or the receptor-binding domain) and those that recognize more than one target. In this regard, a test that considers different epitopes in two separate proteins of the same virus seems to be more sensitive and more appropriate to be used as a screening test.In our single patient experience, this kit was the only one that gave a positive result.It was more sensitive compared to the other similar kits and even more sensitive compared to the rRT-PCR-based methods. The third point concerns the occurrence of lymphocytosis.In our patient, we observed a marked rise in the serum concentration of lymphocytes that was presumably coincident with the onset of COVID-19.It was interpreted as a secondary effect of corticosteroid therapy, and this could be one of the plausible explanations.This finding has been previously reported in other therapy-naive CLL patients with COVID-19 [5].We should consider that lymphopenia is commonly present in patients with COVID-19.However, an opposite behavior of lymphocytes in patients affected by CLL could be remarked.In fact, although only occasionally found, marked lymphocytosis in COVID-19 patients has been described, especially in those also affected by CLL [19].This peak of lymphocytosis might partially mask the occurrence of COVID-19 (as previously suggested [4,5]), which was the case in our patient.However, we cannot exclude that the lymphocytosis may be related to the immune response to the viral infection. The fourth point is the specificity of the clinical presentation and of the radiological images.Both the clinical presentation and the radiological appearance at the HRCT scan of the lung were highly suggestive of the occurrence of COVID-19 in our patient.They represent, in fact, the reason why we kept trying to identify the disease and to reach the diagnosis in our patient. In conclusion, in CLL patients showing a clinical presentation and radiological appearance of the lung suggesting the possible coexistence of COVID-19, the confounding hematological factors that may mask the disease and limit the diagnosis of SARS-CoV-2 infections should be taken into careful consideration. All possible attempts should be made to obtain the correct identification of the disease, using the most accurate methods, to avoid misdiagnosis and delay in identifying the infection, thus preventing the spreading of the virus. ", "section_name": "Discussion and Conclusions", "section_num": "3." } ]	[ { "section_content": "We thank Andrea Smargiassi (Fondazione Policlinico Universitario Agostino Gemelli) for assistance with data collection of the paper. ", "section_name": "Acknowledgments:", "section_num": null }, { "section_content": "Funding: This research received no external funding. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. ", "section_name": "", "section_num": "" }, { "section_content": "Author Contributions: V.V., M.E.L. and S.S. followed the case.G.S., I.S. and M.S. were responsible for laboratory analysis.M.A.V.A.C.L., G.A. and G.L.V. managed the patient during the course of his disease and hospitalization.C.d.D. carried out investigations using diagnostic imaging.V.V. and S.S. supervised the case and S.S. wrote the manuscript.All authors have read and agreed to the published version of the manuscript. The authors declare no potential conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null }, { "section_content": "Author Contributions: V.V., M.E.L. and S.S. followed the case.G.S., I.S. and M.S. were responsible for laboratory analysis.M.A.V.A.C.L., G.A. and G.L.V. managed the patient during the course of his disease and hospitalization.C.d.D. carried out investigations using diagnostic imaging.V.V. and S.S. supervised the case and S.S. wrote the manuscript.All authors have read and agreed to the published version of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare no potential conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.3390/ijms23116260	Venetoclax Induces Cardiotoxicity through Modulation of Oxidative-Stress-Mediated Cardiac Inflammation and Apoptosis via NF-κB and BCL-2 Pathway	<jats:p>Cardiovascular damage induced by anticancer therapy has become the main health problem after tumor elimination. Venetoclax (VTX) is a promising novel agent that has been proven to have a high efficacy in multiple hematological diseases, especially acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). Considering its mechanism of action, the possibility that VTX may cause cardiotoxicity cannot be ruled out. Therefore, this study was designed to investigate the toxic effect of VTX on the heart. Male Sprague-Dawley rats were randomly divided into three groups: control, low-dose VTX (50 mg/kg via oral gavage), and high-dose VTX (100 mg/kg via oral gavage). After 21 days, blood and tissue samples were collected for histopathological, biochemical, gene, and protein analyses. We demonstrated that VTX treatment resulted in cardiac damages as evidenced by major changes in histopathology and markedly elevated cardiac enzymes and hypertrophic genes markers. Moreover, we observed a drastic increase in oxidative stress, as well as inflammatory and apoptotic markers, with a remarkable decline in the levels of Bcl-2. To the best of our knowledge, this study is the first to report the cardiotoxic effect of VTX. Further experiments and future studies are strongly needed to comprehensively understand the cardiotoxic effect of VTX.</jats:p>	[ { "section_content": "Drug-induced toxicity effects on cardiovascular function or tissues are not only a serious health issue, but they are often detected after the introduction of the drug in clinical practice [1].This reflects that these high-risk cardiovascular events are either not detected in earlier clinical trials, or those that arise when drugs are administrated for long periods of time to larger patient population are not considered to be biologically significant [1].Such a high incidence of cardiovascular adverse drug reactions in late-stage clinical development can lead to additional pre-and/or postapproval monitoring, prescribing restrictions, doselimiting toxicity, or ultimately drug discontinuation or withdrawal [1].Importantly, these drug-induced cardiovascular toxicity events are considered the primary cause of drug withdrawal from the market [1,2]. The list of anticancer therapy drugs that can potentially cause cardiotoxicity-related adverse effects is growing [3].This raises an important issue in cancer treatment, as it can influence the mortality and morbidity of patients with cancer by causing a delay or discontinuation of chemotherapy [4].Over the past two decades, anticancer therapy has resulted in remarkable advances in both the survival rate and quality of life of cancer patients [5].Although anticancer agents have shown efficacy against different types of tumors, many reports have demonstrated its cardiotoxic effect [5,6].Cardiotoxicity represents the most feared adverse reaction to chemotherapy, with a growing incidence of up to 30% of patients receiving chemotherapy developing a cardiovascular side effect during their life, which leads to an increase in morbidity and mortality [7][8][9].It is worthy to note that the cardiotoxic side effects induced by long-term use of anticancer therapy has been one of greatest challenges after tumor elimination [10].Therefore, it is essential for oncologists to know the cardiotoxicity profile of newer agents and determine the etiology and most appropriate management of these various effects so they can consider the risks and benefits of eliminating the tumor and preservation of cardiac function [3,11]. Venetoclax (VTX), or ABT-199, is a promising novel agent that has been proven to have a high efficacy in multiple hematological diseases, especially chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) [12].Of all newly diagnosed cancer patients, 6.5% have blood cancers such as acute leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, and multiple myeloma [12].Apoptosis resistance of CLL cells is mediated through Bcl-2 overexpression [13].When Bcl-2 is overexpressed, this leads to tumor formation, as in CLL and follicular lymphoma, inappropriate cell survival, and chemotherapy resistance [14,15].Apoptosis can be initiated by BH3-only proteins in response to significant stresses, such as genetic damage [14].Thus, higher levels of Bcl-2 overcome the BH3-only proteins and result in evading apoptosis [16].VTX targets the BH3 domain of Bcl-2 as a BH3 mimetic (Bcl-2 inhibitor) that can restore apoptosis in malignant cells [17].Indeed, VTX have a high affinity to the BH3-binding groove of Bcl-2, which leads to displacement of the bounded proapoptotic BH3-only proteins [18].Therefore, these free BH3-only proteins lead to displacement and activation of apoptotic effectors (e.g., Bax) from binding to antiapoptotic members [18].Eventually, VTX induces the release of proapoptotic from Bcl-2 and restores apoptosis in tumor cells [18].In the clinical sitting, it has been reported that VTX caused cardiomyopathy and cardiac arrhythmia [19][20][21].Therefore, we hypothesized that VTX treatment can cause toxic effects to the heart.It is well known that the Bcl-2 family of proteins are essential regulators of apoptosis [22].Moreover, it has been reported that inhibition of Bcl-2 can lead to apoptosis in different organs, especially the heart, and consequently resulting in organ toxicity [21,23].Therefore, in the current study, we investigated the toxic effects of VTX on the heart, and examined the signal and molecular mechanism of its toxicity. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "", "section_name": "Results", "section_num": "2." }, { "section_content": "It has been demonstrated that cardiac enzymes increase in blood when there are some injuries or damage to the heart.Therefore, we measured the serum level of CK-MB and cardiac troponin I (cTn-I) in rats after 21 days of VTX treatment.Surprisingly, we found that the treatment of VTX increased the serum levels of both CK-MB (Figure 1A) and cTn-I (Figure 1B), but the increase in cTn-I was not significant.These results indicated that VTX treatment induced cardiac damage. ", "section_name": "Effect of VTX Treatment on Cardiac Enzymes", "section_num": "2.1." }, { "section_content": "Numerous studies have shown that cardiac hypertrophy can be a maladaptive process in response to intrinsic or extrinsic stresses.Therefore, we measured the body weight (BW), heart weight (HW) and heart weight/body weight ratio (HW/BW), which we used as an indicator of cardiac hypertrophy, and measured the expression of three genes that are known to be involved in this process.As shown in Figure 2A,B, no significant changes were observed in the BW or HW between all groups.However, a significant increase in the HW/BW ratio was observed in VTX-treated groups compared to the control group (Figure 2D).Furthermore, we found that the gene-expression level of α-Mhc decreased in VTX-treated groups compared to the control group, while the gene-expression levels of β-Mhc and the β-Mhc to α-Mhc ratio increased significantly in the VTX-treated groups compared to the control group (Figure 2A-C, respectively).Furthermore, the gene-expression level of Bnp was substantially increased in the VTX-treated groups (Figure 2D).Overall, these results indicated that VTX treatment induced cardiac hypertrophy and damage. G) Bnp were measured using RT-PCR.Data are presented as mean ± SD (n = 5).* p < 0.05, ** p < 0.01; n.s., no significant changes were observed (p > 0.05).Data were normalized to β-actin as a housekeeping gene and one-way analysis of variance (ANOVA), followed by Tukey-Kramer multiple-comparisons tests.Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg); α-Mhc, alpha myosin heavy chain; α-Mhc, beta myosin heavy chain; Bnp, brain natriuretic peptide. ", "section_name": "VTX-Induced Cardiac Hypertrophy", "section_num": "2.2." }, { "section_content": "It has been demonstrated that changes in and damage to cardiac cell morphology correlate with many diseases and toxicity.Therefore, we examined the heart architecture of rats after 21 days of VTX treatment.We observed a normal and parallel myocardial fiber with cross-striation and regular nuclei in the myocardium sections obtained from the control group (Figure 3A,D).Sections of myocardium obtained from the second group, which received a low dose of VTX, also showed a normal appearance of the myocardial fibers, but with minimal nuclear enlargement (Figure 3B,E).Interestingly, heart sections from the third group, which received a high dose of VTX, showed the presence of a focus of myocardial damage associated with chronic inflammatory reaction (arrowhead) (Figure 3C,F).Figure 3F shows the arrowhead area at 600× magnification, and indicates the presence of a focus of subendocardial myxoid degeneration.This feature is not well understood, but could indicate myocardial damage in this area.Furthermore, we observed that VTX treatment increased the cardiomyocytes' cross-sectional area considerably in a dose-dependent pattern (Figure 3H).Taken together, VTX treatment induced morphological changes in cardiac tissues. ", "section_name": "Effects of VTX on Cardiac Architecture", "section_num": "2.3." }, { "section_content": "Several studies have linked myocardial dysfunction and toxicity with apoptosis.Therefore, we measured the gene and protein expressions of multiple apoptotic markers to examine the induction of apoptosis in heart tissues.We found that 21 days of VTX treatment induced the gene expression of Bax compared to the control group (Figure 4A).Moreover, a significant decline was observed in the Bcl-2 gene and protein expressions in VTX-treated rats compared to the control group (Figure 4B,C, respectively).Nonetheless, a Western blot analysis revealed that treatment with a high dose of VTX resulted in a notable rise in the protein levels of cleaved caspase-3 (Cleaved Cas-3) compared to the control group (Figure 4D). ", "section_name": "VTX-Induced Apoptosis in the Heart", "section_num": "2.4." }, { "section_content": "Cas-3 protein levels.Data are presented as mean ± SD (n = 5).* p < 0.05, ** p < 0.01; n.s., no significant changes were observed (p > 0.05).Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg); Bax, Bcl-2 associated X; Bcl-2, B-cell lymphoma 2; Cleaved Cas-3, cystinyl aspartate-specific proteases 3; β-actin, beta actin. ", "section_name": "(D) Representative Western blot analysis of Cleaved", "section_num": null }, { "section_content": "It has been demonstrated that oxidative stress and inflammation are the main mechanisms that induce cardiac toxicity.Therefore, we measured the gene and protein expressions of different inflammatory and oxidative stress markers.We found that the gene-expression levels of Ifn-γ (Figure 5A) and Tgf-β (Figure 5B) sharply rose in the VTX treatment group compared to the control group.Furthermore, the gene and protein levels of Nf-κb-p-65 in VTX-treated rats were significantly increased in the high-dose VTX group compared to the control group (Figure 5C,F, respectively).Moreover, the levels of gene and protein expressions of Tnf-α (Figure 5D,G, respectively) and Il-6 (Figure 5E,H, respectively) were remarkably increased in the treatment groups compared to the control group in a dosedependent manner.Strikingly, the level of the antioxidant protein, Sod-2, significantly declined in rats treated with a high dose of VTX compared to the control group (Figure 5I).Taken together, these results provided important insight regarding the involvement of inflammation and oxidative stress in cardiac toxicity in VTX treatment.(A-E) The mRNA levels of Ifn-γ, Tgf-β, Nf-κb-p-65, Tnf-α, and Il-6 were measured using RT-PCR.Data were normalized to β-actin as a housekeeping gene and one-way analysis of variance (ANOVA), followed by Tukey-Kramer multiple-comparisons tests.(F-I) Representative Western blot analysis of protein levels of Nf-κb-p-65, Tnf-α, Il-6, and Sod-2.Data are presented as mean ± SD (n = 5).* p < 0.05, p < 0.01, * p < 0.001; n.s., no significant changes were observed (p > 0.05).Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg); Ifn-γ, interferon gamma; Tgf-β, transforming growth factor beta; Nf-κb-p-65, nuclear factor kappa-B; Tnf-α, tumor necrosis factor alpha; Il-6, interleukin-6; Sod-2, superoxide dismutase-2; β-actin, beta actin. ", "section_name": "VTX-Induced Oxidative Stress and Inflammation in the Heart", "section_num": "2.5." }, { "section_content": "ROS production is involved in the cardiotoxicity of many anticancer drugs.It can cause oxidative damage to many vital components of the cell, including DNA and proteins, as well as mitochondrial dysfunction and cell death.It had been found that increased levels of lipid peroxidation led to an increase in cardiotoxicity.Moreover, decreased levels of antioxidants in the heart have been associated with the production of ROS and cardiotoxicity.Therefore, we measured the levels of malondialdehyde (MDA), catalase (CAT), and glutathione (GSH).We found that the VTX-treated groups had increased levels of MDA compared to the control group (Figure 6A).Moreover, the levels of CAT (Figure 6B) and GSH (Figure 6C) were remarkably diminished in both VTX-treatment groups compared to the control group.In summary, these results confirmed that VTX treatment induced oxidative stresses. ", "section_name": "Effect of VTX on Oxidative Stress Status", "section_num": "2.6." }, { "section_content": "In the present study, we investigated whether VTX treatment could induce toxic effects in the heart.We found that VTX treatment induced cardiotoxicity that was manifested by changes in the histological architecture of cardiomyocytes, an increase in cardiac enzymes and the expression of relevant genes of cardiac injury, induction of apoptosis markers, alterations in oxidative stress markers, and an increase in inflammatory markers. VTX is a recently approved anticancer drug that is used for the treatment of multiple hematological cancers [12].VTX acts as a selective inhibitor of the BH3 domain of Bcl-2 that can restore apoptosis in cancer cells [17].In the clinical sitting, it has been reported that VTX caused cardiomyopathy and cardiac arrhythmia [19][20][21].Therefore, we hypothesized that VTX treatment could cause toxic effects to the heart.It is well known that the Bcl-2 family of proteins are essential regulators of apoptosis [22].Moreover, it has been reported that inhibition of Bcl-2 can lead to apoptosis in different organs, resulting in organ toxicity [21,23].However, to date, there are no preclinical reports that investigated the cardiotoxicity of VTX.Therefore, in the current study, we investigated the toxic effects of VTX on the heart, and examined the signal and molecular mechanisms of its toxicity. Cardiac enzymes, such as myocardial muscle creatine kinase (CK-MB) and cardiac troponin (cTn-I), are key tools for evaluating heart damage, as well as histopathological examination of cardiomyocytes, which have been found to be a major marker of the cardiotoxic effect of anticancer drugs [24,25].Furthermore, alterations in the enzymatic reaction, such as CK-MB and cTn, represent a key early response to toxicant exposure in the heart [26].In the present study, we found that levels of both CK-MB and troponin I had increased significantly in the VTX-treated rats compared to the nontreated controls.These results indicated that VTX treatment was associated with cardiomyocyte damage.Moreover, our histopathological findings in rats that received VTX treatment showed the presence of minimal nuclear enlargement and focus of myocardial damage associated with a chronic inflammatory reaction.Moreover, we showed that VTX treatment substantially increased the size of cardiac myocytes in a dose-dependent manner.These histopathological findings also confirmed that VTX treatment caused cardiomyocytes injuries. Cardiac hypertrophy is thought to be a maladaptive compensatory mechanism of the heart in response to toxic insult [27].Changes in cardiac hypertrophic markers such as Bnp, α-Mhc, and β-Mhc have been found to be associated with cardiac toxicity, and were reported for several anticancer drugs, such as doxorubicin and sunitinib [28,29].In the current study, we found that VTX treatment increased the gene expression of β-Mhc, Bnp, and the β-Mhc:α-Mhc ratio compared to the control group, while α-Mhc gene expression was decreased.These results suggested a VTX-mediated hypertrophy and toxicity on the heart. Cardiomyocyte death is considered the main cause of cardiotoxicity [30].The most common form of cell death in drug-induced cardiotoxicity is apoptosis [30,31].Many cytotoxic insults, such as DNA damage and oxidative stress, can activate the intrinsic apoptotic pathway, which is controlled by the Bcl-2 family of proteins [32,33].This family can be divided into proapoptotic and antiapoptotic members.The Bcl-2 protein is present in the outer mitochondrial membrane, and acts as an antiapoptotic protein by preventing the release of cytochrome c into the cytosol.On the other hand, intrinsic cell apoptosis can also be regulated by Bax, a proapoptotic member of the Bcl-2 family that can cause cytochrome c release and activate multiple caspases, eventually leading to cell death.Thus, the balance between Bcl-2 and Bax can influence the cell survival or death [23,30,34].In this study, we found that 21 days of treatment with VTX led to cardiomyocyte death, as evidenced by reduced levels of the gene and protein expressions of Bcl-2, increased levels of Bax gene expression, and elevated cleaved caspase-3 (Cleaved Cas-3) protein expression levels. It is well known that cardiomyocyte death induced by ROS production is involved in many cardiac pathological conditions, such as cardiac hypertrophy and HF [35,36].Moreover, overproduction of oxygen free radicals and induction of oxidative stress can lead to chemokine production, recruitment of inflammatory cells, and activation of transcription factors such as Nf-κb [36].Inflammation as a result of oxidative stress is associated with a plethora of pathological diseases, including diabetic cardiomyopathy, congestive cardiomyopathy, and hypertensive heart disease [37].Increased levels of cytokines in the blood or cardiomyocyte have been found in diseases that lead to cardiomyocyte death.Nf-κb is a transcription factor that upregulates the production of downstream inflammatory mediators, including Tnf-α, Il-6, Ifn-γ, and Tgf-β [38].Moreover, Ifn-γ and Il-1 are both proinflammatory cytokines that can induce Tnf-α production by cardiomyocytes [38].Nevertheless, Il-6, which is a proinflammatory cytokine, was found to be elevated in patients with heart failure [26].Tnf-α is one of the inflammatory mediators that has an important role in the induction of myocardial cell apoptosis [26].Furthermore, Tgf-β plays an important role in apoptosis, wound healing, and immune regulation [39].It has been reported that Tgf-β overexpression was associated with fibrosis and hypertrophy [40].Our findings revealed that 21 days of treatment with VTX resulted in ROS production and inflammation that led to apoptosis of cardiomyocytes, which was confirmed by the increase in the gene and protein expression of Nf-κb-p-65 and the decrease in the Sod-2 protein level.Additionally, the induction of Ifn-γ gene levels, as well as Tnf-α and Il-6 gene-and protein-expression levels, further confirmed the toxic consequences of VTX in the heart.Lastly, increased Tgf-β gene-expression levels confirmed our histopathological findings, in that VTX treatment induced maladaptive cardiac hypertrophy and eventually cardiac damage. To further confirm the oxidative stress production, we measured the levels of malondialdehyde (MDA), catalase (CAT), and glutathione (GSH) activity.MDA is a lipid peroxidation end-product and a gold standard marker of lipid peroxidation and oxidative stress.Moreover, GSH and CAT are key endogenous antioxidants that are critical to maintaining cellular homeostasis and ROS levels in response to different toxic insults [41,42].In this study, we observed a significant dose-dependent reduction in GSH and CAT in response to VTX treatment.Furthermore, our results also demonstrated a significant dosedependent increase in MDA levels in the VTX-treated groups compared to the control group.These results confirmed our previous findings that VTX treatment induced oxidative stress in the heart. ", "section_name": "Discussion", "section_num": "3." }, { "section_content": "", "section_name": "Materials and Methods", "section_num": "4." }, { "section_content": "Male Sprague-Dawley rats weighing 180-200 g were used.The rats were obtained from Prince Naif Bin AbdulAziz Health Research Center, King Saud University, Riyadh, Saudi Arabia.All experiments on rats were conducted according to the standard guidelines and approved by the Animal Care and Use Committee at King Saud University, Saudi Arabia (Approval# KSU-SE-19-86).Animals were housed under normal laboratory conditions (temperature of 25 ± 1 • C) of a 12 h light/dark cycle with free access to water and a normal chow diet. ", "section_name": "Animals", "section_num": "4.1." }, { "section_content": "Rats were arbitrarily divided into three groups, with 8 rats in each group.These groups were as follows: Group 1: Rats in this group were treated daily via oral gavage with 0.9% NaCl for 21 days, and it served as the control group. Group 2: Rats in this group were treated daily with a low dose of VTX (50 mg/kg) via oral gavage for 21 days (LDV). Group 3: Rats in this group were treated daily with a high dose of VTX (100 mg/kg) via oral gavage for 21 days (HDV). VTX doses were selected based on previous published studies [43][44][45][46][47].For all groups, rats were weighed daily to calculate the dose and monitored for any changes in weight, as well as for any signs of toxicity.On day 21, rats were anesthetized using ketamine 100 mg/kg and xylazine 10 mg/kg intraperitoneally [48].Blood samples were collected from all rats.Then, the serum was separated for further measurement of cardiac enzymes.Consequently, heart tissues were harvested and washed twice with ice-cold phosphatebuffered saline; some tissues were fixed in formaldehyde solution (4%) for histopathology studies, while other tissues were stored at -80 • C to conduct different biochemical, gene, and protein studies.The ratio of heart weight to body weight (HW/BW) was used as an indicator of myocardial mass, as described previously [49,50]. ", "section_name": "Study Design", "section_num": "4.2." }, { "section_content": "Coagulated blood was centrifuged at 2000× g for 10 min at 4 • C to separate the serum from the whole blood.Serum creatine kinase MB isoenzyme (CK-MB) and cardiac troponin I (cTn-I) were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) (ABBEXA, Cambridge, UK) as per the manufacturer's protocol. ", "section_name": "Measuring the Cardiac Enzymes", "section_num": "4.3." }, { "section_content": "Total cellular RNA was isolated from the heart tissues by using TRIzol reagent (Ambion, Austin, TX, USA) following the manufacturer's protocol.The isolated RNA was assessed by using a NanoDrop™ 8000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) to verify the quality and quantity.Then, the isolated RNA (1 µg) was reverse transcribed to cDNA by using a reverse transcription kit (BIMAKE, Houston, TX, USA).Gene expressions were quantified by using the appropriate primers listed in Table 1, a 7500 Fast Real-Time PCR system (ThermoFisher Scientific, Waltham, MA, USA), and SYBR green master mix (BIMAKE, Houston, TX, USA).Data were normalized to β-actin as a housekeeping gene.The forward and reverse primers used are listed in Table 1.The data were acquired during the extension step.All primers were obtained from Integrated DNA Technologies (IDT, Leuven, Belgium).The DDCt method was used to calculate the relative levels of mRNA expression [51]. ", "section_name": "Gene Expression Studies", "section_num": "4.4." }, { "section_content": "A Western blot was used to determine the protein levels of Nf-κb-p-65, Sod-2, Cleaved Cas-3, Bcl-2, Tnf-α, and Il-6.In brief, previously collected heart tissues were homogenized using Dounce homogenizer in ice-cold RIPA lysis buffer (ThermoFisher Scientific, Waltham, MA, USA) that was supplemented with protease and phosphatase cocktail inhibitor (Ther-moFisher).After that, an equal amount of proteins (25-50 µg) were electrophoresed using SDS-PAGE, then transferred onto a PVDF membrane.Following the transfer, membranes were blocked for one hour at room temperature with 5% nonfat dry milk with gentle rocking.Membranes were incubated overnight in the primary antibody at 4 • C with gentle rocking (dilution at 1:1000).These primary antibodies were rabbit anti-nuclear factor kappa B-p-65 (Nf-κb-p-65) antibody, rabbit anti-superoxide dismutase-2 (Sod-2) antibody, rabbit anti-cleaved caspase-3 (Cleaved Cas-3) antibody, rabbit anti-B-cell lymphoma-2 (Bcl-2) antibody, rabbit anti-tumor necrosis factor (Tnf-α) antibody, rabbit anti-interleukin 6 (Il-6) antibody, and mouse anti-B-actin antibody.Thereafter, membranes were incubated for one hour at room temperature with the appropriate horseradish peroxidase (HRB) conjugated secondary antibody (dilution at 1:5000) (ABclonal, Wuhan China).Finally, membranes were visualized using chemiluminescence reagent (Millipore, Burlington, MA, USA) and imaged using a Bio-Rad gel-imaging system (Bio-Rad, Hercules, CA, USA). ", "section_name": "Protein-Expression Studies", "section_num": "4.5." }, { "section_content": "Lipid peroxidation was measured in cardiac tissues by adding thiobarbituric acid (TBA) and trichloroacetic acid (TCA) to tissue homogenates.Then, this mixture was incubated for 30 min in a shaking water bath at 90 • C.Then, the samples were placed on ice for 10 min.Thereafter, the samples were centrifuged for 15 min at 3000× g in a refrigerated centrifuge, and the supernatant was measured at 540 nm.The values of results were expressed in nmol of MDA formed per mg of protein [52]. ", "section_name": "Measurement of Lipid Peroxidation", "section_num": "4.6." }, { "section_content": "The amount of GSH in tissues was measured using a previously described method [53].In brief, 5,50-dithio bis (3-nitrobenzoic acid) was added to the reaction mixture.Then, the absorbance was immediately recorded at 412 nm.The values of GSH were expressed as nmol/mg of protein. ", "section_name": "Measurement of Reduced Glutathione", "section_num": "4.7." }, { "section_content": "The postmitochondrial supernatant (PMS) from heart tissue was used to estimate the CAT activity by using a previously described method [54].In brief, the reaction mixture, in a total volume of 3 mL, consisted of 1.95 mL (0.1 M, pH 7.4) phosphate buffer, 1 mL (0.019 M) hydrogen peroxide, and 0.05 mL PMS.The absorbance was recorded for 5 min at 240 nm at an interval of 1 min.To calculate the activity of CAT, the difference in the absorbance was used as the amount of moles of H2O2 changed per min per mg of protein. ", "section_name": "Measurement of Catalase Activity", "section_num": "4.8." }, { "section_content": "Heart tissues from all groups were collected, fixed in 4% formaldehyde, and embedded in paraffin.Then, thin 3 mm sections were prepared using a microtome and stained with hematoxylin and eosin (H&E) to examine the heart morphology.The morphology of the cardiac cells and the nucleus of myocardial fiber cells were compared using an optical microscope to evaluate the severity of cardiac damage (Olympus BX microscope and DP72 camera, Melville, FL, USA).The damage quantification from at least 10 areas corresponding to the myocardial tissue was graded using the following parameters: nuclear enlargement and inflammation based on a four-score evaluation system (0, histopathological changes = 1-25%; 1, histopathological changes = 26-50%; 2, histopathological changes = 51-75%; and 3, histopathological changes =76-100%).This procedure was conducted in at least 10 random areas in each heart section, in three animals from each group, at 400× magnification.The mean score for each parameter was calculated and subjected to statistical analysis.The cardiomyocyte cross-sectional size was estimated and evaluated using the H&E stained slides. ", "section_name": "Histopathology", "section_num": "4.9." }, { "section_content": "All data are presented as mean ± SD and analyzed using GraphPad Prism version 6.01 (San Diego, CA, USA).Different results between groups were analyzed using a oneway or two-way analysis of variance (ANOVA), followed by a Tukey-Kramer multiplecomparisons test with significance values of p < 0.05. ", "section_name": "Statistical Analysis", "section_num": "4.10." }, { "section_content": "To the best of our knowledge, this was the first study to report that VTX treatment could induce cardiotoxicity in a dose-dependent manner.This cardiotoxicity caused by VTX treatment was manifested in different ways, including modification or changes in the histological architecture of cardiomyocytes, increases in cardiac enzymes such as CK-MB and troponin I, and alteration of cardiac hypertrophic genes markers such as Bnp, α-Mhc, and β-Mhc (Figure 7).Our findings revealed that VTX treatment induced apoptosis in cardiac tissues as a result of Bcl-2 reduction and Cleaved Cas-3 and Bax induction.Moreover, increased levels of MDA in cardiac tissues and reduced levels of GSH, CAT, and Sod-2 caused oxidative stress that led to activation of Nf-κb-p-65 and induction of the inflammatory response, which was observed as the increases in the expressions of Ifn-γ Tgf-β, Tnf-α, and Il-6.The results of this study add to the current knowledge regarding the safe use of VTX.One of the limitations of the present study was that we did not use knockout models to inhibit the molecular mechanisms involved in VTX-induced cardiotoxicity.Furthermore, we did not measure any in vivo parameters, such as those from echocardiography or blood pressure measurements, which could help to further assess the effects of VTX on heart function.However, the results of the current study shed light on the toxic effects of VTX treatment on the heart, and encourage future studies to further prove the current findings.Further studies are required to fully and comprehensively understand the exact mechanism of VTX-induced cardiotoxicity. ", "section_name": "Conclusions", "section_num": "5." } ]	[ { "section_content": "The authors are thankful to the Researchers Supporting Project (number RSP-2021/335), King Saud University, Riyadh, Saudi Arabia. ", "section_name": "Acknowledgments:", "section_num": null }, { "section_content": "Funding: This project was funded by the Researchers Supporting Project (number RSP-2021/335), King Saud University, Riyadh, Saudi Arabia. Board Statement: The animal study protocol was approved by the KSU Local Institutional Study Ethics Committee (REC) (protocol code KSU-SE-19-86, 2019).Informed Consent Statement: Not applicable. ", "section_name": "Institutional Review", "section_num": null }, { "section_content": "Funding: This project was funded by the Researchers Supporting Project (number RSP-2021/335), King Saud University, Riyadh, Saudi Arabia. ", "section_name": "", "section_num": "" }, { "section_content": "Board Statement: The animal study protocol was approved by the KSU Local Institutional Study Ethics Committee (REC) (protocol code KSU-SE-19-86, 2019).Informed Consent Statement: Not applicable. ", "section_name": "Institutional Review", "section_num": null }, { "section_content": "Data Availability Statement: Data are contained within the article. ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare no conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.1158/0008-5472.22394210.v1	Supplementary Figures 1-5 from Extensive Promoter DNA Hypermethylation and Hypomethylation Is Associated with Aberrant MicroRNA Expression in Chronic Lymphocytic Leukemia	<jats:p><p>PDF file - 90K, Supplementary Figure S1 contains technical validations of differentially methylated miRNA promoters by high resolution quantitative DNA methylation analysis. Supplementary Figure S2 shows experimental validation of promoter activity for the mir-129-2 locus. Supplementary Figure S3 shows relative expression of predicted miRNA target genes by qPCR in CLL specimens and healthy B cell controls. Supplementary Figure S4 shows an unsupervised clustering of quantitative DNA methylation data at the identified hyper- and hypomethylated miRNA promoters in CLL specimens, healthy B cell samples, various normal hematopoietic cells and several solid tissues. Supplementary Figure S5 shows a detailed quantitative DNA methylation analysis of the miR-155 host gene 5' region and correlations with miR-155 expression in CLL specimens, healthy B cell samples, various normal hematopoietic cells and several solid tissues</p></jats:p>	[ { "section_content": "", "section_name": "", "section_num": "" } ]	[ { "section_content": "Results are depicted as heatmap (yellow, unmethylated; blue, highly methylated).(C) Average DNA methylation over the depicted regions is correlated with expression as measured by qPCR in the respective tissue relative to the average expression of the three houskeeping RNAs SCARNA17, SNORD25 and SNORA73A.For CLL, healthy B and T cells as well as granulocytes, the average of all respective samples is used. ", "section_name": "", "section_num": "" } ]
10.3390/cells10020217	Nurse-Like Cells and Chronic Lymphocytic Leukemia B Cells: A Mutualistic Crosstalk inside Tissue Microenvironments	<jats:p>Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries and is an example of hematological disease where cooperation between genetic defects and tumor microenvironmental interaction is involved in pathogenesis. CLL is a disease that is considered as “addicted to the host”; indeed, the crosstalk between leukemic cells and the tumor microenvironment is essential for leukemic clone maintenance supporting CLL cells’ survival, proliferation, and protection from drug-induced apoptosis. CLL cells are not innocent bystanders but actively model and manipulate the surrounding microenvironment to their own advantage. Besides the different players involved in this crosstalk, nurse-like cells (NLC) resemble features related to leukemia-associated macrophages with an important function in preserving CLL cell survival and supporting an immunosuppressive microenvironment. This review provides a comprehensive overview of the role played by NLC in creating a nurturing and permissive milieu for CLL cells, illustrating the therapeutic possibilities in order to specifically target and re-educate them.</jats:p>	[ { "section_content": "Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in Western countries and predominantly affects the elderly, with a median age at diagnosis of 72 years.CLL is characterized by the accumulation of clonal mature CD5 + B cells in peripheral blood, bone marrow, and secondary lymphoid organs.The diagnosis of CLL is established by testing the presence of ≥5 × 10 9 /L monoclonal B lymphocytes in the peripheral blood.In the absence of lymphadenopathy, organomegaly, cytopenia, and clinical symptoms, the presence of <5 × 10 9 /L monoclonal B lymphocytes defines \"monoclonal B lymphocytosis\" (MBL), which progresses to CLL in at least 1-2% of MBL cases per year [1]. From a biological and clinical perspective, CLL displays a wide degree of heterogeneity, ranging from patients characterized by a stable disease with a nearly normal life expectancy to patients with an aggressive disease with frequent relapses or transformation into an aggressive lymphoma, such as diffuse large B-cell lymphoma (DLBCL) (Richter transformation).Patients affected by CLL have rising lymphocytosis, adenopathy, hepatosplenomegaly, and bone marrow infiltration, resulting in bone marrow failure with anemia and thrombocytopenia [2].The reason for such clinical heterogeneity has not been completely elucidated; it is mainly determined by genetic factors and the complex relationship that leukemic cells have with the surrounding microenvironment.CLL is characterized by a distinct immunophenotype with the co-expression of CD19, CD5, and CD23 and low levels of surface immunoglobulins.Studies conducted on the B-cell receptor (BCR) have led to identifying two molecular subgroups: those harboring unmutated immunoglobulin heavy-chain variable region (IGHV) genes (U-CLL, ≥98% identity with the germline) and those with mutated IGHV genes (M-CLL).U-CLL originates from B cells that have not experienced the germinal center reactions, whereas M-CLL originates from post-germinal center B cells [3].Unmutated IGHV genes predict worse prognosis, and this is possibly due to the enrichment of some genetic lesions that confer high aggressiveness (such as mutations of NOTCH1) and also the predisposition of U-CLL to undergo clonal evolution [2].In addition, U-CLL cells show a higher rate of in vitro spontaneous apoptosis and are more dependent on the surrounding environment as compared to CLL cells isolated from M-CLL patients [4].Noteworthy, the BCR of almost 30% of CLL patients displays nearly identical or highly homologous complementarity determining region 3 (CDR3), also known as \"stereotyped\" BCR, suggesting an antigen-driven path to CLL development.Of note, this feature of stereotypy implies shared somatic mutations, a similar genetic profile of the leukemic clone, and also similar antigen-binding properties and functional responses through the BCR and similar clinical outcomes [5]. Historically, CLL has been considered as an accumulative disease of B lymphocytes with defects in apoptosis that morphologically resemble small resting B cells in the blood with limited ability to proliferate [3,6].Nowadays, the dynamic nature of CLL clones has been defined, showing the cellular proliferation of leukemic clones inside structures called proliferation centers or pseudofollicles frequently found in the lymph nodes and bone marrow of CLL patients [7,8].In pseudofollicles, specific cellular populations, including T cells, stromal cells, and macrophages, in addition to the endothelial cells and B prolymphocytes/paraimmunoblasts, are present.Inside these structures, CLL cells' survival and proliferation is preserved through the activation of BCR signaling [9]. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "In 1889, Stephen Paget stated that \"when a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil\", proposing that the microenvironment provides a fertile \"soil\" for tumor cells (\"seeds\"), leading to their growth [10]. Evidence of microenvironment dependency is related to the knowledge that despite an apparent long life in vivo, CLL cells derived from peripheral blood die spontaneously and rapidly when cultured in in vitro conditions with media supplemented with either autologous or fetal bovine serum [11].This suggests that extrinsic signals from microenvironmental elements surrounding leukemic cells in vivo are essential to support prolonged CLL cell survival.In bone marrow and secondary lymphatic tissues, CLL cells entertain complex cellular and molecular interactions with the surrounding accessory cells, collectively referred to as the \"microenvironment\" [12].Comparison between different anatomic compartments of the disease, such as peripheral blood, bone marrow, and lymph nodes, has underlined the importance of the crosstalk between leukemic cells and bystander non-malignant cells in lymphoid organs to support CLL maintenance [13].Noteworthy, lymphocytes continually recirculate from blood to tissues and back to the bloodstream again.Trafficking is mediated by transient interactions with the endothelium through adhesion molecules and chemokines that trigger integrin activation, thus inducing firm adhesion and transendothelial migration into tissues where stromal cells guide lymphocyte homing and retention [14].In CLL, trafficking and homing into the lymph nodes and bone marrow are an essential part of the disease pathogenesis and progression.Inside tissue niches, CLL cells establish a tight and intimate interaction with many cell types such as extracellular matrix, fibroblasts, cells of the innate and adaptive immune response, and vascular endothelial cells (ECs).This bidirectional network is guaranteed by cell-to-cell contact, adhesion molecules, cell surface ligands, chemokines, cytokines, and their corresponding receptors [15].CLL cells are not innocent bystander but actively model and manipulate the surrounding microenvironment to their own advantage.For this reason, it is thought Cells 2021, 10, 217 3 of 12 that the CLL microenvironment is shaped and maintained through a dynamic, interactive coevolution between leukemic and normal accessory cells [12].The main players inside tissue microenvironments are mesenchymal stromal cells, leukemia-related macrophages, T cells, NK cells, and endothelial cells.This complex crosstalk is crucial for CLL cells' survival and proliferation signals and makes a critical contribution to disease progression and drug resistance or disease relapse [16][17][18][19][20][21]. Different studies have focused the attention on the influence of bone marrow stromal cells in preventing the apoptosis of CLL cells.Adhesive interaction between them is able to deliver signals which regulate the survival pathways in CLL cells, inducing protection from spontaneous apoptosis and reducing susceptibility to therapeutic agents [22,23].Among the plethora of factors involved in this crosstalk, VLA-4 integrin (CD49d), on CLL cells plays a critical role through the binding to VCAM-1 (CD106) on the stromal cell surface.VLA-4 is variable expressed by CLL patients and predicts disease progression; indeed, high levels of VLA-4 are associated by a more disease progression [24,25].Binding of VCAM-1 by VLA-4 is able to confer protection to CLL cells from apoptosis and drug resistance [26].Again, CD40 is expressed on CLL cells and is able to bind its ligand, CD40L, that is found on activated T lymphocytes.In CLL, the triggering of CD40 induces the survival and proliferation of leukemic cells, counteracting the therapeutic effects of apoptogenic drugs [27][28][29]. Leukemia-cell derived extracellular vesicles represent another possibility of communication with the surrounding microenvironment.Exosomes contain functionally active biological molecules, such as proteins, lipids, mRNA, and microRNA.These molecular structures deliver their content along the bloodstream and lymphatic vessels and release it into the target cells influencing their cellular functions.In CLL, it has been demonstrated that exosomes are able to rescue CLL cells from spontaneous and drug-induced apoptosis, enhance their migration ability, and support the proliferation of leukemic cells [30,31]. BCR activation is critical for CLL maintenance, as its signal transduction pathway is essential for CLL cells' survival, proliferation, and trafficking.Antigen binding to the BCR activates proximal kinases such as SYK and LYN along with phosphorylation of immunoreceptor tyrosine-based motifs receptor of Igα/Igβ.This activates SYK, BTK, PI3Ks, calcium mobilization, MAP/kinases, phospholipase-C-γ, and NF-κB [32,33].In particular, BTK is a member of tyrosine protein kinase (Tec) with a critical role in the amplification of the BCR signal.The activation of BTK is involved in the PI3K/Akt pathway and also in the activation of Iκb kinase, which phosphorylates the NF-κB inhibitor I-κBα kinase, allowing NF-κB to translocate to the nucleus [34,35].The interaction of CLL cells with the tumor microenvironment is controlled by BCR signaling and is involved in the survival and proliferation of leukemic cells [36]. As mentioned, CLL cells are not passive seeds but are able to create supportive conditions aberrantly orchestrating the function of immune effector cells and escaping immunosurveillance.Indeed, CLL cells directly participate to immunomodulation supporting an immunosuppressive environment.The formation of a CLL-induced immunosuppressive milieu includes molecular mechanisms, environmental influence and immune evasion [37].This is in line with the observation that CLL is characterized by several clinical complications related to alterations in the immune system, including hypogammaglobulinemia, predisposition to infection, and the increased incidence of autoimmune disorders and secondary primary malignancies [38]. ", "section_name": "Role of Microenvironment in CLL", "section_num": "1.1." }, { "section_content": "Inside tissue niches, monocyte/macrophage population assumes a critical role in the maintenance and progression of CLL cells.CLL patients have a high number of circulating monocytes with significant shift toward non-classical population with low level of CD14 together with high CD16 [39].Non-classical monocytes exhibit patrolling behavior in vivo, are weak phagocytes, and do not produce cytokines in response to cell-surface toll-like receptors [40].The altered composition and function of blood monocytes in CLL patients could derive from a specific CLL-mediated education of immune cells including an establishment of a skewed phenotype in the monocyte/macrophage population.In tissues infiltrated by CLL cells, it has been identified a specific population of leukemia-associated macrophages called nurse-like cells (NLC).NLC own their name, given important similarities with thymic nurse cells (TNC) that reside in thymic cortex.TNC is a large epithelial cell that contains many viable lymphoid cells within its intracellular vesicles and it provides a microenvironment that is necessary for lymphocyte proliferation and differentiation.Inside TNC, lymphocytes are morphologically intact, and many of them undergo mitosis without any sign of phagocytic degradation.Lymphocytes roll under these cells without been internalized according to a process known as emperipolesis [41,42].The first observation on the formation of NLC in CLL is thanks to work carried out by Burger et al [43].The isolation and culture of peripheral mononuclear cells (PBMC) in complete medium for at least 14 days lead to formation of peculiar adherent cells, now known as NLC.(Figure 1) Formation of NLC is closely related to PBMC isolated from CLL patients, indeed culture of PBMC from healthy donors does not lead to formation of NLC unless establishment of a co-culture of CD14 + cells of healthy donors with CLL B cells.This strongly suggests the influence of leukemic cells in the generation of NLC.NLC form a monolayer of large, round, and sometimes binucleate cells, with CLL cells firmly attached all around them [43].NLC have an immunophenotype that distinguishes them from blood circulating monocytes, monocyte-derived dendritic cells, or macrophages.They express at different levels CD14, CD45, HLA-DR, CD33, and CD68 and fail to express CD106, which is usually observed on follicular, dendritic cells marrow and stromal cells.Evidence of NLC' existence in vivo derives from the culture of CLL splenocytes, which allow the growth of cells very similar to NLC, suggesting that cells with distinctive properties of NLC are present in the secondary lymphoid tissues of CLL patients [18].In the microenvironment, CLL cells may drive the induction of NLC differentiation through several factors.The enzyme nicotinamide phosphoribosyltransferase (NAMPT) is expressed at high levels by CLL cells and this is also confirmed by measuring extracellular NAMPT at elevated amounts in the plasma of CLL patients.Extracellular NAMPT produced by CLL cells has a pivotal role in polarizing circulating monocytes into macrophages, and blocking NAMPT results in compromising NLC differentiation and phenotype [44].Moreover, high-mobility group protein B1 (HMGB1) is a nuclear protein that is usually released by damaged cells or dead cells or by immune cells and cancer cells [45].It has been demonstrated that CLL cells are able to release HMGB1, regulating NLC differentiation through TLR-9/RAGE pathway [46].Colony-stimulating factor-1 is required for normal homeostasis and survival of macrophages.Mice lacking functional CSF-1 or nullizygous for CSF-1 receptor have a decrease in tissue-resident macrophages [47].NLC express the CSF-1 receptor, which is important for their generation and survival [48]. ", "section_name": "Nurse Like Cells", "section_num": "1.2." }, { "section_content": "The increased number of NLC in the CLL microenvironment has been associated with disease progression and shorter overall survival [46,49].The complex interplay between CLL cells and NLC inside tissue niches leads to the protection of CLL cells promoting their recruitment, survival, and proliferation.NLC express high levels of stromal-derived factor 1-α (SDF-1α), a chemokine that is a potent chemoattractant for CLL cells involved in leukemic cells' migration and emperipolesis [50].SDF-1α secreted by NLC drives CLL cells inside the protective tissue niches through the activation of the corresponding CXCR4 receptor expressed on the surface of CLL cells.Once leukemic cells are attached to NLC, they are protected from apoptosis through a firm cell-to-cell contact [51].The engagement of CXCR4 in CLL cells by SDF-1α induces the activation of downstream signaling pathways as MAP kinase and AKT, crucial in the maintenance of CLL cells' survival [43,52].In addition, NLC express B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) that are both TNF family members, with critical roles in peripheral B cell survival, maturation, and differentiation.BAFF binds to three separate receptors expressed by CLL cells, the BAFF receptor (BAFF-R), the transmembrane activation and calcium modulation ligand interaction (TACI), and the B cell maturation antigen (BCMA).APRIL binds only TACI and BCMA [53].The stimulation of these receptors induces the activation of NF-κB pathway and the expression of Bcl-2, preserving CLL cells' survival [54].In the same line, brain-derived neurotrophic factor (BDNF), secreted by NLC, activates the complex NTSR2-TrkB expressed at the CLL cells' surface, inducing Src pro-survival signal and the expression of Bcl-2 [55].Again, NLC release galectin 1 (Gal1) into the surrounding environment.Gal1 is found in the inflammation sites and tumor growth, performing prosurvival effects on malignant cells.Gal1 influences CLL behavior through the modulation of BCR signaling, decreasing the threshold of its activation, or through the control of BAFF and/or APRIL secretion determining opportune microenvironmental conditions for leukemic progression [56].In response to antigen(s) presented by NLC, BCR activation triggers leading to the increased secretion of chemokines CCL3 and CCL4 by CLL [57].These chemokines mobilize accessory cells, such as T cells and monocytes, to tissue niches, allowing the formation of a nurturing milieu for CLL cells [58].As confirmation, gene expression profile analyses conducted on peripheral blood, bone marrow, and lymph nodes samples have demonstrated an inducible, probably antigen-drived BCR signaling in CLL cells in the lymph node that has resulted in Syk and NF-κB activation and the induction of a characteristic gene expression signature [13].In the CLL microenvironment, secreting and stimulating factors are only partial culprits in the maintenance of leukemic cells' survival.It has to be considered that CLL cells adhere closely to NLC, establishing firm physical contact.The contact of CLL cells with NLC preserves leukemic cells' viability through the interaction of LFA-3 (lymphocyte function-associated antigen 3), broadly expressed in leukemic cells that bind CD2 on NLC.The LFA-3/CD2 axis exerts a pro-survival effect on CLL clones and of note LFA-3 expression correlates with increased overall survival after frontline rituximab-based immunochemotherapy [59]. ", "section_name": "Nurturing Properties of NLC", "section_num": "1.3." }, { "section_content": "The increased number of NLC in the CLL microenvironment has been associated with disease progression and shorter overall survival [46,49].The complex interplay between CLL cells and NLC inside tissue niches leads to the protection of CLL cells promoting their recruitment, survival, and proliferation.NLC express high levels of stromal-de- ", "section_name": "Nurturing Properties of NLC", "section_num": "1.3." }, { "section_content": "Cells of the monocyte-macrophage lineage are characterized by diversity and plasticity.In response to different signals, macrophages may be skewed toward classical M1 activation or alternative M2 activation.In particular, M1 polarization is characterized by the expression of high levels of pro-inflammatory cytokines, the production of reactive nitrogen and oxygen intermediates, the promotion of Th1 response, and strong microbial activity.M2 polarized-macrophages promote tissue remodeling and tumor progression and have immunoregulatory functions [60].CLL patients are characterized by high number of circulating Tie-2-expressing monocytes (TEMs).TEMs are highly proangiogenic subset of myeloid cells in tumors and display immune suppressive activity [61].In addition, Tie2 + NLC are detected in CLL-infiltrated lymph nodes, mainly in a peri-vascular distribution, suggesting that leukemic cells secreting Ang2 in infiltrated-tissue recruit them into tissue from the TEM subpopulation [39,62]. NLC have been considered as CLL-specific tumor-associated macrophages, showing the modulation of genes involved in the regulation of both innate and acquired immunity with impairment in the phagocytic capability [63].Accordingly, CD163, a marker of macrophages with M2 polarization, is widely expressed in NLC.Its function is carried out through the scavenging of the haptoglobin-hemoglobin complex and the production of anti-inflammatory metabolites [64].It has been established that the expression of CD163 by NLC correlates with CLL proliferation in lymph nodes and the soluble counterpart of CD163 links with worst prognostic factors, such as TP53 mutations, complex karyotype.and unmutated IGHV.In addition, high levels of CD163 are associated with shorter overall survival and treatment-free survival [49].The gene expression profiling of NLC shows the dysregulation of genes involved in immunocompetence, with a high expression of CD11b, CD68, CD206, IL-10, CCL-18, and IDO.NLC and CLL monocytes inhibit T-cell proliferation through TGFβ, IL-10, and IDO; in addition, soluble factors produced by NLC drive the expansion of T regulatory cells, reinforcing a link between the impairment of immune response and CLL progression [44,65,66]. ", "section_name": "Immunosuppressive Properties of NLC", "section_num": "1.4." }, { "section_content": "In the last few years, it has been delineated that NLC do not just nurse but actively participate in the setting of environment-mediated drug resistance and immunosuppression.For this reason, different studies have investigated the rationale of targeting NLC in order to modify their nurturing and protective behavior, looking towards the induction of an immunological re-education (Figure 2).Liposomal clodronate is widely used for the in vivo depletion of macrophages; studies conducted in CLL mice have shown an impairment of CLL development, the resolution of immune dysfunction, and a partial resolution of systemic inflammation [67]. Given the importance of CSF-1 receptors in macrophages' survival, the inhibition of CSF1-R signaling leads to macrophage depletion in CLL mice, determining their reduction in bone marrow and peripheral blood.Macrophages, targeted via the CSF-1 receptor, shift the CLL microenvironment phenotype toward a more anti-tumor direction, preventing the formation of new macrophages by inducing apoptosis or affecting macrophage differentiation from monocyte precursors and reducing the leukemic cell load [68,69]. Trabectedin is an antitumor agent of marine origin extracted from the tunicate Ecteinas cidia turbinata.It acts as a minor groove of the DNA binder that blocks the cell cycle, affects gene transcription, and alters the DNA repair pathway.Trabectedin shows selective cytotoxic effects on monocytes and macrophages, affecting monocyte differentiation to macrophages [70].In CLL, trabectedin has shown to induce cytotoxic effects in leukemic cells and concomitantly exerts immunomodulatory activity on several cell types of the microenvironment.It depletes myeloid-derived suppressor cells and tumor-associated macrophages and induces T cell response [71].Given the importance of CSF-1 receptors in macrophages' survival, the inhibition of CSF1-R signaling leads to macrophage depletion in CLL mice, determining their reduction in bone marrow and peripheral blood.Macrophages, targeted via the CSF-1 receptor, shift the CLL microenvironment phenotype toward a more anti-tumor direction, preventing the formation of new macrophages by inducing apoptosis or affecting macrophage differentiation from monocyte precursors and reducing the leukemic cell load [68,69]. Trabectedin is an antitumor agent of marine origin extracted from the tunicate Ecteinascidia turbinata.It acts as a minor groove of the DNA binder that blocks the cell cycle, affects gene transcription, and alters the DNA repair pathway.Trabectedin shows selective cytotoxic effects on monocytes and macrophages, affecting monocyte differentiation to macrophages [70].In CLL, trabectedin has shown to induce cytotoxic effects in leukemic cells and concomitantly exerts immunomodulatory activity on several cell types of the microenvironment.It depletes myeloid-derived suppressor cells and tumor-associated macrophages and induces T cell response [71]. Lurbinectedin, a trabectedin analogue, is a selective inhibitor of protein-coding genes, leading to arrest the elongation of RNA polymerase II and its degradation by the ubiquitin/proteasome machinery.The recruitment of DNA repair factors determines an Lurbinectedin, a trabectedin analogue, is a selective inhibitor of protein-coding genes, leading to arrest the elongation of RNA polymerase II and its degradation by the ubiquitin/proteasome machinery.The recruitment of DNA repair factors determines an accumulation of double-strand breaks, with consequent apoptosis.In CLL, lurbinectedin has a direct effect on leukemic cells and also on the monocyte/macrophage population.This drug indirectly reduces the number of NLC interfering with CLL cells homing in proliferation centers.In addition, lurbinectedin increases the production of IL-1β by monocytes and NLC that is associated with good prognostic markers or increased survival [72].In order to reprogram NLC, a possibility is the use of interferon γ (IFN-γ).Interferons are considered modulators of macrophage plasticity and activation, in particular, IFN-γ is involved in the promotion of monocyte differentiation.The treatment of NLC with IFN-γ modulates them toward a more effector-like phenotype, concomitantly interfering with CLL cell survival.In the presence of IFN-γ, NLC function as immune effectors with decreased M2 features and increased phagocytic ability [73]. Lenalidomide is an immunomodulatory drug that has been used in CLL.It acts not directly on CLL cells but by modulating the tumor microenvironment.Lenalidomide affects the protection of leukemic cells induced by NLC, inhibiting the migration of CLL cells toward SDF-1α [74].Furthermore, lenalidomide counteracts the ability of leukemic cells to generate NLC.Instead, it promotes the expansion of a macrophage population with the M1 phenotype characterized by enhanced phagocytic activities and supports T-cell proliferation, with less ability to nurture leukemic cells.Lenalidomide is able to decrease Ibrutinib is an irreversible inhibitor of BTK that has demonstrated exceptional efficacy in CLL patients [76,77].Ibrutinib disrupts the BCR and NF-κB pathways, affecting CLL trafficking, homing, and viability.Despite the impressive improvement in clinical outcomes, some patients show limited benefit from BTK inhibition due to discontinuation due to adverse effects related at least in part to off-target effects related to the tumor microenvironment [78].Ibrutinib is not able to mobilize NLC from tissues into the bloodstream and supports the protection of NLC to promote CLL survival [79].Ibrutinib exerts its immunomodulatory effects on NLC through the modulation of BTK expression in this population.BTK in macrophages is involved in the regulation of lineage commitment and the inhibition of BTK in NLC, potentiating the M2-skewed features.Ibrutinib induces the inhibition of phagocytosis, the induction of of M2 markers such as CD163 and CD206, together with negative regulation of M1 polarization; and modulates clusters of genes involved in immune suppression [80]. Recently, invasive fungal infections have been reported among patients receiving treatment with ibrutinib.In this scenario, ibrutinib is able to affect the ability of NLC to counteract Aspergillus fumigatus conidia germination due to reduced phagocytosis and the impairment of a productive inflammatory response with a decreased level of IL-1β and TNF-α [81,82]. ", "section_name": "NLC as a Therapeutic Target", "section_num": "1.5." }, { "section_content": "CLL is the most common form of adult leukemia in the Western countries.Besides a dynamic landscape of genetic alterations, the disease history is related to the complex intimate crosstalk that leukemic cells entertain with non-malignant accessory cells inside tissue microenvironments.Despite several and important therapeutic advances, nowadays CLL is still an incurable disease and clinical resistance may occur both through the primary biological features of tumor cells or through resistance, which arises through the crosstalk with the surrounding tumor microenvironment.Inside tissue niches, nurse-like cells represent the Achilles' heel in CLL.In this scenario, NLC are not just caregiver of CLL cells, feeding and protecting them from drugs, but given the complex bidirectional crosstalk, NLC are manipulated and programmed by leukemic cells in order to create an immunosuppressive milieu that allows immune evasion.Therapeutic strategies to directly target NLC, blocking their formation or re-educating these cells against leukemia, need to be envisioned. ", "section_name": "Conclusions", "section_num": "2." } ]	[ { "section_content": "Funding: This work was supported by Associazione Italiana per la Ricerca sul Cancro (IG21436 R.M.), Progetto Dipartimenti di Eccellenza 2018-2022.S.F. is supported by an annual fellowship from Fondazione Umberto Veronesi, Italy. ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare no conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.18632/oncotarget.12097	NK cell function is markedly impaired in patients with chronic lymphocytic leukaemia but is preserved in patients with small lymphocytic lymphoma	Chronic lymphocytic leukemia (B-CLL) and small lymphocytic lymphoma (SLL) are part of the same disease classification but are defined by differential distribution of tumor cells. B-CLL is characterized by significant immune suppression and dysregulation but this is not typical of patients with SLL. Natural killer cells (NK) are important mediators of immune function but have been poorly studied in patients with B-CLL/SLL. Here we report for the first time the NK cell phenotype and function in patients with B-CLL and SLL alongside their transcriptional profile. We show for the first time impaired B-CLL NK cell function in a xenograft model with reduced activating receptor expression including NKG2D, DNAM-1 and NCRs in-vitro. Importantly, we show these functional differences are associated with transcriptional downregulation of cytotoxic pathway genes, including activating receptors, adhesion molecules, cytotoxic molecules and intracellular signalling molecules, which remain intact in patients with SLL. In conclusion, NK cell function is markedly influenced by the anatomical site of the tumor in patients with B-CLL/SLL and lymphocytosis leads to marked impairment of NK cell activity. These observations have implications for treatment protocols which seek to preserve immune function by limiting the exposure of NK cells to tumor cells within the peripheral circulation.	[ { "section_content": "Chronic lymphocytic leukemia (B-CLL) and small lymphocytic lymphoma (SLL) have identical morphological and immunophenotypic features on tissue biopsy and are grouped together within the World Health Organization classification of hematological malignancy (WHO 2008) [1].B-CLL is defined by peripheral blood lymphocytosis, bone marrow infiltration, and a variable degree of lymph node expansion [2,3].In contrast, patients with SLL do not demonstrate lymphocytosis and have a low incidence of cytopenias secondary to bone marrow infiltration [2].The mechanisms underlying this differential distribution of tumor cells are not known but are likely to relate to fundamental properties of the tumor cell, such as chemokine receptor expression [4].SLL is approximately ten times less common than B-CLL and accounts for 6% of all Non-Hodgkin lymphoma [5]. B-CLL is associated with a range of immunological complications including an increased propensity to infection and auto-immune complications [3,6,7].In contrast, these complications are not a major feature of SLL [8] and most likely reflect differences in the tissue distribution of the tumor cells rather than the overall tumor load.A number Research Paper of immunological abnormalities have been described in patients with B-CLL including hypogammaglobulinaemia and T cell dysfunction [9].Natural killer cells (NK cells) are innate lymphocytes that play a critical role in the immune surveillance against infection and malignancy and also contribute to the therapeutic activity of current treatment protocols for B-CLL/SLL [10].However, the phenotype and function of NK cells in patients with B-CLL and SLL have been relatively poorly investigated.Tumors evolve many mechanisms to evade elimination by NK cells [11,12], and defects in the cytotoxic capacity of NK cells from patients with B-CLL were described as early as 1996 [13].The mechanisms that underlie this phenotype remain uncertain but are thought to reflect an alteration in the balance between activatory and inhibitory signaling [14][15][16]. We investigated the in vitro and in vivo function of NK cells from patients with B-CLL and SLL and observed a selective and marked functional impairment in cells taken from patients with B-CLL.Global downregulation of several activating receptors, including NKG2D, DNAM-1 and NCRs, was observed on NK cells from patients with B-CLL.Using whole genome transcription microarray of NK cells, the transcription of many genes involved in cytotoxic function was also found to be dysregulated.These data reveal a profound and selective impairment of NK cell function in patients with B-CLL compared to those with SLL.The differential distribution of the B-CLL/ SLL tumor within blood is therefore a critical determinant of NK cell function.These data are relevant to the potential detrimental influence of lymphocytosis during 'watch and wait' clinical monitoring or during treatments with targeted therapies that mobilize tumors cells into the bloodstream. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "In order to investigate the functional capacity of NK cells taken from patients with B-CLL, an in-vitro cytotoxicity assay was carried out using the NK cell target line K562 [17].NK cells were isolated from healthy donors (HD-NK) or patients with B-CLL (CLL-NK) prior to incubation with CFSE-labeled K562 cells.43% of target cells were lysed following incubation with HD-NK cells (mean ± SEM: 43% ± 3.5%) but this was reduced by 40% following incubation with CLL-NK (mean ± SEM: 25.8% ± 2.6; p = 0.0017) (Figure 1A).This result has been confirmed by using Europium release based cytotoxicity assay (Supplementary Figure S1).In contrast, NK cells from patients with SLL demonstrated no significant difference in their lytic capacity compared to NK cells from HD (mean ± SEM: 41.7% ± 4.9; P = 0.56) (Figure 1A). In order to assess how this impairment in vitro function was translated into activity in vivo we next used a xenograft model of NK cytotoxicity.NOG mice were injected subcutaneously with K562 cells and then at day 3 NK cells, from either HD or patients with B-CLL, were infused.IL-2 was given to support NK cell expansion and a control group of mice received IL-2 treatment alone.K562 tumor growth became apparent in all mice at day 7 after injection and tumor size was measured on day 10, 14 and 17 (Figure 1B).NK cells taken from HD substantially reduced the growth of the K562 tumor such that tumor volume was suppressed by 54% at day 17.Tumor sizes derived from control mice were 1910 ± 290 mm3 (mean ± SEM) compared to 890 ± 200 mm3 in those mice infused with HD-NK cells (p = 0.029) (Figure 1C).In contrast, NK cells taken from patients with B-CLL were incapable of any significant degree of tumor suppression (Figure 1C). ", "section_name": "NK cells from patients with B-CLL demonstrate functional impairment during assays of in vitro and in vivo activity", "section_num": null }, { "section_content": "NK cell cytotoxicity is mediated through a range of activating receptors, of which NKG2D-mediated signaling is a dominant pathway.As such, we next went on to determine the surface expression of NKG2D on NK cells taken from HD and patients with B-CLL (n = 23).A markedly reduced expression of NKG2D was observed on NK cells from patients with B-CLL but not SLL, in comparison to the profile on cells from HD (Figure 2A).In particular, the percentage of NKG2D-positive NK cells was reduced by 51% amongst patients with B-CLL (mean ± SEM B-CLL 43.1% ± 2.7% vs HD 86.6% ± 2.7%; p < 0.001; Figure 2B).Interestingly, the percentage of NKG2D positive NK cells was not reduced in patients with SLL (mean ± SEM 85.3% ± 2.9%) in comparison to that observed on NK cells from HD (Figure 2B). In order to assess if this reduction of NKG2D surface expression on NK cells from patients with B-CLL is functionally significant we then determined the ability of NK cells to mediate NKG2D-dependent cytotoxicity.Specifically, a CHO cell line was stably transfected with ULBP6, an NKG2D ligand, and was used as a target cell, whilst untransfected CHO cells served as a negative control.The cytotoxicity to this cell line has been shown to be able to be blocked by anti-NKG2D antibody (Supplementary Figure S4).NKG2D-mediated killing was substantially reduced using NK cells from patients with B-CLL in comparison to either NK cells taken from HD or patients with SLL.In particular, specific lysis of the ULBP6expressing CHO cell line by NK cells from HD was 47% compared to only 29% using NK cells taken from patients with B-CLL (mean ± SEM: HD 47% ± 7.0% vs B-CLL, 29% ± 5.4% p < 0.05) (Figure 2C).Again, NK cells taken from patients with SLL did not reveal any impairment of lytic activity compared to HD (mean ± SEM SLL 48.5% ± 6.1%) (Figure 2C). ", "section_name": "NKG2D expression and NKG2D-mediated cytotoxic function are both decreased in NK cells taken from patients with B-CLL but not SLL", "section_num": null }, { "section_content": "In addition to NKG2D, several other activating receptors are expressed on NK cells, including DNAM-1 and the natural cytotoxicity receptors (NCR) NKp30 and NKp46.The surface expression of DNAM-1, NKp30 and NKp46 was examined on NK cells taken from HD and patients with B-CLL or SLL.All three activating receptors were markedly downregulated on NK cells from patients with B-CLL compared to NK cells from HD.Of note, the percentage of DNAM-1 positive NK cells was 89.3% ± 1.1% on HD-NK compared to 75.5% ± 2.9% on CLL-NK cells (p < 0.001) (Figure 3A left panel).Similarly, the percentage of NKp46 positive NK cells was 58.3% ± 3.3% on HD-NK compared to 35.4% ± 3.2% on CLL-NK cells (p < 0.001) (Figure 3A middle panel).Also the percentage of NKp30 positive NK cells was 73.6% ± 4.2% and 61.1% ± 4.0% (p = 0.0152) on HD-NK and CLL-NK cells respectively (Figure 3A right panel).In contrast, the percentage of these receptors positive NK cells from patients with SLL was not significantly different from those found on HD-NK (DNAM-1, 86.0% ± 2.3%; NKp46, 59.2% ± 6.7%; NKp30, 68.0% ± 8.3%) (Figure 3A).Together, these data show that expression of activating receptors is substantially reduced on NK cells taken from patients with B-CLL, but not from patients with SLL.Also the mean fluorescence intensity (MFI) of surface expression of NKG2D, DNAM-1 and NCRs showed similar trend of reduction on NK cells from patients with B-CLL, but not from patients with SLL (Supplementary Figure S3). ", "section_name": "The expression of DNAM-1 and natural cytotoxicity receptors are also reduced on NK cells from patients with B-CLL but not SLL", "section_num": null }, { "section_content": "The Fc receptor, CD16, is an activating receptor expressed on the majority of NK cells and plays an important role in mediating ADCC.The pattern of CD16 expression was next examined on NK cells from patients with B-CLL and SLL (gating strategy shown in Supplementary Figure S5).75.7% ± 3.5% of NK cells from HD were CD16 bright , compared to only 39.0% ± 5.3% of NK cells derived from patients with B-CLL (p < 0.001) (Figure 3B).The percentage of CD16 bright NK cells in patients with SLL (79.1% ± 2.9) was not different to HD-NK cells (Figure 3B). ADCC is an important effector mechanism underlying the activity of rituximab, a CD20-specific monoclonal antibody used in the management of B-CLL [18].In order to assess if the reduction in the proportion of CD16 bright NK cells from patients with B-CLL might impact on rituximab efficacy, a rituximabsensitized ADCC in vitro assay was undertaken, with primary allogeneic B-CLL tumor cells used as targets.In the absence of rituximab, killing of B-CLL tumor cells was relatively modest regardless of the source of NK cells, reflecting natural resistance of primary B-CLL tumor cells to NK cell mediated lysis.However, even here tumor cell killing was reduced using NK cells from patients with B-CLL (2.2% ± 1.3) compared to NK cells from HD (15.7% ± 3.2) or patients with SLL (23.4% ± 4.0) (Figure 3C).The addition of rituximab led to a substantial increase in tumor lysis by all three NK effector subgroups (they are all allogeneic NK cells against a single clone of primary tumor cells).However, although tumor killing by CLL-NK cells increased by nearly 20-fold following rituximab, from 2.2% to 43%, this was still significantly lower than the lysis observed with rituximab in combination with NK cells from HD and patients with SLL (69% and 58% respectively) (Figure 3C).Similar results has been obtained using the enriched B cells from healthy donors as target cells (Supplementary Figure S6).These results indicate that allogeneic NK cells from patients with B-CLL demonstrate reduced lysis of primary B-CLL cells, which is partially but not completely, enhanced in the setting of ADCC. ", "section_name": "NK cells from patients with B-CLL express lower levels of CD16 and display impaired antibodydependent cell mediated cytotoxicity (ADCC)", "section_num": null }, { "section_content": "To investigate the transcriptional basis for the functional and phenotypic impairment of NK cells from patients with B-CLL, we used GeneChip ® Human Transcriptome Array 2.0.and performed comparative transcriptional profiling of NK cells taken from HD or patients with either B-CLL or SLL (n = 5 in each group).70523 positive probe-sets were found among the whole chip-set. The pattern of gene expression within NK cells from patients with B-CLL or SLL was compared to the transcriptional profile observed in NK cells taken from HD. 480 and 667 genes showed at least a 1.5 fold change in transcript levels within CLL-NK and SLL-NK samples respectively compared to HD-NK (p < 0.05).However, it was noteworthy that the great majority of these differences were not shared between patients with SLL and B-CLL.In particular, 331 genes were downregulated in CLL-NK samples and 189 genes were downregulated within the SLL-NK group.However, only 46 of these genes shared a common pattern of downregulation within both groups (Figure 4A middle panel, Figure 4B top panels).A further 149 and 478 genes demonstrated increased expression within CLL-NK and SLL-NK samples respectively, whilst only 36 genes were upregulated in both patient groups (Figure 4A right panel, Figure 4B bottom panels).Together, 93 shared genes were significantly modified within both groups compared to HD-NK (Figure 4A left panel).46 of these shared genes were downregulated, 36 were upregulated in both groups and 11 differed in their direction between the two groups.DAVID functional annotation tool was then used to identify gene sets of GO (Gene ontology terms) that were enriched.Consistent transcription changes were observed within each sample of the three participant groups.The 93 genes included genes encoding phosphoproteins (38 genes), regulation of transcription (15 genes), intracellular signaling cascade (11 genes) and regulation of apoptosis (9 genes) (Figure 4A left panel and Supplementary Figure S7). ", "section_name": "The transcriptional profiles of NK cells from patients with either B-CLL or SLL display a unique profile", "section_num": null }, { "section_content": "We next determined the biochemical pathways that are modulated by the pattern of trancriptional expression within NK cells.KEGG pathway analysis (http://www.genome.jp/kegg/pathway.html)highlighted the gene group responsible for NK cell-mediated cytotoxicity that were downregulated by more than 1.5 fold within CLL-NK samples compared to HD-NK (p < 0.05).These were then grouped into four classes, based on the NK cytotoxic pathway (Figure 5B), namely activating receptors (NCR1, NCR3, FCGR3A, FCGR3B, FCER1G, HCST), adhesion molecules (NCAM1, ITGAL, ITGB2, SPN, ITGAM), cytotoxicity molecules (GZMA, GZMB, GZMK, PRF1) and genes involved in the cytotoxic intracellular signaling cascade (NFATC2, SYK, VAV1, RAC2, PRKCA, PRKCB, LAT2) (Figure 5A).These data indicate that several biochemical pathways important for cytotoxic funcion are imparied in NK cells from patients with B-CLL. The profile of these genes was next examined in NK cells taken from patients with SLL and, with the exception of VAV1 which was reduced by 48% in patients with SLL, all genes were found to be expressed at the same level as cells from HD (Figure 5A).These data are consistent with the substantial difference in the pattern of functional cytotoxicity observed between NK cells taken from patients with B-CLL or SLL. In order to assess how the transcriptional profile correlated with the pattern of surface expression, we next correlated microarray data with flow cytometric analysis.The relative transcription levels for NCR1 (NKP46) and NCR3 (NKp30) were 94 and 81 in HD-NK, 60 and 45 in CLL-NK and 123 and 93 in SLL-NK.As described above, these levels were significantly lower in CLL-NK cells compared to HD-NK and SLL-NK (P = 0.001 for NCR1 and 0.0002 for NCR3) with no difference observed between HD-NK and SLL-NK.Interestingly, flow cytometric analysis of surface expression of NKp46 and NKp30 revealed the same pattern, with MFI of 402 and 2650 respectively in HD, 337 and 2060 in patients with B-CLL and 432 and 2240 in patients with SLL (Supplementary Figure S3).A further 5 cytotoxic-related genes, (NCAM1 (CD56), FCGR3A (CD16a), FCGR3B (CD16b), PRF1 (perforin) and GZMB (granzyme B), also exhibited a strong correlation between transcriptional profile and surface or intracellular protein expression (data not shown). Interestingly, the level of NKG2D transcription did not differ between CLL-NK and HD-NK cells.However, NKG2D is expressed as a dimer associated with the adaptor protein DAP10 [19,20] and expression of the HCST gene, which encodes DAP10 was markedly reduced in CLL-NK cells compared with HD-NK and SLL-NK (mean transcription: HD-NK 240 vs CLL-NK 162 vs SLL-NK 428 respectively) (Figure 5A).The association with DAP10 is a pre-requisite for NKG2D expression at the cell surface [21] and DAP10 phosphorylation is required for the intracellular signaling that mediates NK cell activation [22,23].As such, the reduction in DAP10 transcription in CLL-NK cells is sufficient to account for the observation of decreased NKG2D expression and impaired NKG2Dspecific killing by CLL-NK cells (Figure 2). ", "section_name": "Multiple genes involved in NK cytotoxicity are expressed at lower level in NK cells from patients with B-CLL", "section_num": null }, { "section_content": "NK cells have a critical role in the control of infection and malignant disease [24], but have been relatively poorly investigated in patients with B-CLL, despite the observation that both of these complications are increased in this disorder.Our study has revealed significant functional impairment of NK cells from patients with B-CLL and used for the first time transcriptional and flow cytometric analysis to reveal multiple mechanisms in NK cytotoxicity impairment.Additionally, we show that NK cell cytotoxicity was unimpaired in patients with SLL, a clinical disorder that is defined as part of the same disease entity as B-CLL. Impaired cytolytic function of NK cells in patients with B-CLL has previously been described [13,16,25,26].This impairment has been attributed to a lack of azurophilic granules, impaired release of cytolytic molecules [13], and impaired expression of activating receptors [16].In agreement with others, our study has confirmed the impaired cytotoxic function of NK cells from patients with B-CLL in vitro using K562 target cells but have further extended this observation in vivo using a xenograft model.Importantly, we also for the first time use an NKG2D-dependent cytotoxicity assay to demonstrate that reduced NKG2D expression directly correlated with impairment in NK cell cytotoxicity (Figure 2C). Our microarray analysis contrasted the transcriptional profile of NK cells from HD or patients with either B-CLL or SLL and remarkably found that many genes, which regulate cytotoxicity are downregulated in NK cells taken from patients with B-CLL.These included NK activating receptors such as NKp30, NKp46 and several adhesion molecules.Adhesion molecules are required for immunological synapse formation, which has been reported to be impaired in patients with B-CLL and improves with the use of lenolidamide [27].We also found significant reduction in expression of genes involved in cell signalling and cytotoxic granule formation.The combination of these defects would all contribute to the impaired cytotoxic function observed in NK cells from patients with B-CLL. NK cells are believed to be important in the elimination of tumor cells.It is therefore not surprising that tumors evolve mechanisms to evade elimination by NK cells, including secretion of immunoregulatory molecules such as PGE2 and TGF-β [28,29].Confirming the findings of others, we incubated HD-NK cells with TGFβ-1 for 48hrs and found the surface expression of NKG2D was reduced by 53% (p = 0.014) (Supplementary Figure S8) [30,31].And also B-CLL tumor cells produce considerable amounts of TGF-β1, such that elevated levels are found in patient serum [32,33].Soluble TGF-β1 therefore represents one mechanism by which the functional impairment of NK cells in patients with B-CLL may arise. One of our most striking and novel observations was the profound difference in the profile of NK cell populations taken from patients with B-CLL or SLL.Indeed, NK cells from patients with SLL showed no significant impairment in function or phenotype compared to cells taken from HD.The clinical difference between B-CLL and SLL is the tissue distribution of the tumor cells.SLL is characterized by lymphadenopathy and/or splenomegaly in the absence of a peripheral lymphocytosis, whereas B-CLL is defined by the presence of tumor cells in the blood.As such, NK cells in patients with B-CLL will be exposed continually to tumor cells within the circulatory system where chronic signaling interactions, such as tumor cell expressing and shedding NKG2D and NCR ligands, will lead to NKG2D downregulation and potential NK 'exhaustion' [14,34].Of note we did not find any difference in the level of soluble NKG2D ligands within serum between HD and patients with B-CLL (Supplementary Figure S9).Interestingly, a murine model of NK cell adoptive transfer has also demonstrated the rapid down-regulation of activating receptors in a pattern that correlated with tumor exposure [35].An overview of potential mechanisms is shown in Figure 6. Our observations further suggest that NK cells do not interact significantly with tumor cells within lymph nodes in patients with SLL.During health, the great majority of NK cells circulate within the blood and demonstrate a cytolytic CD56 dim phenotype.However a population of CD56 hi NK cells is observed within lymph nodes where they differentiate from a CD34 dim CD45RA + hematopoietic precursor cell under the influence of IL-12 or IL-15 [36][37][38].We performed immunohistochemical analysis to determine NK cell expression in lymph node sections taken from patients with SLL (n = 6) or B-CLL at stage B disease (n = 2).Interestingly, CD56 + or CD16 + NK cells were completely absent in tumor lymph nodes, a pattern that was in marked contrast to the profile observed in healthy lymph node sections and has not previously been reported.No differences were observed between lymph node sections of SLL and B-CLL. GO (Gene ontology terms) analysis of the microarray result from DAVID functional annotation tool showed that there were 32 genes involved in cell migration, cell motion and chemokine signalling pathway are significantly modulated in B-CLL patients group compared with HD (Supplementary Figure S10).7 genes, VAV1, PRKCA, EPHA4, SELPLG, ID1, MYH9 and CXCR3, were consistently modulated in both the B-CLL and SLL groups.Several of these proteins are involved in mediating signaling pathways that activate the actin cytoskeleton and cell migration.Two particularly interesting genes are CXCR3 and SELPLG (CD162) which play an important role in the migration of NK cells between the lymphoid compartment and peripheral tissues [39].Together our data indicate that NK cells are largely unable to enter lymph nodes containing B-CLL/SLL tumor cells and that this helps to preserve peripheral NK cell function in patients with SLL. These data suggest that the function of the immune system is significantly less impaired in patients with SLL compared to those with B-CLL.Interestingly there has been relatively little study of comparative immune function and relative infection risk in patients with these two disorders.Reduced levels of IgM and IgG are observed in patients with SLL although the IgA concentration is well preserved [40], a factor that has been recently correlated with reduced infection risk in patients with B-CLL who have been treated with ibrutinib [41].In contrast, the frequency of auto-immune hemolysis and thrombocytopenia is markedly less common in SLL but previous work has not found any evidence of difference in the T cell compartments between the two disease subsets [8,42].Although there are no epidemiological studies of the incidence of infectious complications, the widespread clinical impression is that these are less common in patients with SLL, and indeed other patients with low grade non Hodgkins lymphoma, than in patients with B-CLL.To our knowledge this work represents the first comparison of NK cell function from patients with SLL and B-CLL and suggests that preservation of NK cell function could play an important role in maintaining immune homeostasis in this disorder. Strikingly, the changes described in this work were identified in patients with early stage B-CLL, in whom a 'watch and wait' approach to treatment is almost universally adopted.The findings add to the knowledge that clinical observation of early stage B-CLL is associated with gradual impairment of immune function [43] and may contribute to arguments in favour of the early control of the tumor clone.Indeed, downregulation of NKG2D on NK cells has been correlated with disease progression [16] but no such association was observed in our cohort due to the relatively early clinical stage of the patient group.The data are also of interest in relation to the use of signal transduction inhibitors, such as ibrutinib or idelalisib, which can induce a state of lymphocytosis for several months after treatment and which might therefore lead to further impairment of NK function.This suggests that combination therapy with agents such as anti-CD20 antibodies, which control drug-induced lymphocytosis, may help to support NK cell function.However, further studies are needed in this regard and information to date has suggested that infectious complications and immune dysfunction are actually reduced in patients who are treated with these agents [41].Our data suggests that interventions to improve NK cell function could be a promising strategy to improve the clinical outcome for patients with B-CLL.Indeed, it has been suggested that some of the clinical efficacy of lenalidomide in B-CLL derives from its ability to increase the expression of NKp30 on NK cells and increase tumor cell lysis [44].In addition recombinant IL15 has also been shown to be presented by B-CLL tumor cells to NK cells in vitro where it leads to NK activation and subsequent tumor cell lysis [45]. In summary, we have demonstrated a profound functional impairment of cytotoxic activity in NK cells from patients with B-CLL and have shown for the first time using transcription profiling, that this arises as a result of a global impairment of several biochemical pathways within the cytotoxic machinery.Additionally, we show this pattern is not present in patients with SLL, indicating that NK cell function is determined primarily by the anatomical site of the tumor clone.This observation has significant implications for the development of future clinical protocols, which seek to optimize immune function in the management of B-CLL. ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "Patients with B-CLL were recruited from clinics at Birmingham Heartlands Hospital and University Hospital Birmingham over a 6 month period.Patients were assessed by an experienced Hematologist and fulfilled IWCLL criteria for B-CLL or WHO criteria for SLL.23 Patients with B-CLL were confirmed to be untreated and Binet stage A at recruitment, with a median age of 70 years (IQR 63-81).8 untreated Patients with SLL were also studied, with a median age of 70 years (IQR 59.8-76.3)and either palpable or CT evidence of lymphadenopathy which was histologically proven to be SLL, in the absence of a peripheral lymphocytosis.All patients provided written informed consent in accordance with ethical approval obtained from the local regional ethics committee (REC no 10/H1206/58).For comparative analysis, 23 healthy age-matched donors were recruited as part of the ongoing 1000 elders recruitment at University of Birmingham, which recruits local healthy individuals who are over the age of 65.Their median age was 73 years (IQR 67-83.2).Written informed consent was obtained and 50 ml of peripheral blood donated (REC no 2002/073). ", "section_name": "Participants", "section_num": null }, { "section_content": "Following all blood donations, peripheral blood mononuclear cells (PBMCs) were extracted over a ficoll density gradient and stored at -160 o C. PBMCs were later defrosted and used for functional NK cell studies, immunophenotyping and NK cell enrichment prior to microarray analysis. After defrosting, PBMCs were washed and re-suspended at 10 6 cells/100 μl before staining with live/ dead ® red stain dye (Invitrogen; Massachusetts, USA).Cells were subsequently washed before incubation with one of the following antibody panels: NKG2D panel; anti-γδ PE, anti-CD3 FITC (Beckman Coulter, USA); anti-CD8 Amcyan, anti-NKG2D APC (BD bioscience, USA), anti-CD4 Percp-Cy5.5 (eBioscience, USA) anti-CD56 PE-Cy7 (Biolegend, USA).DNAM panel; anti-γδ PE; anti-CD8 Amcyan, anti-CD4 Percp-Cy5.5,anti-CD56 PE-Cy7, anti-DNAM FITC, (Biolegend) anti-CD3 APC-Cy7 (BD bioscience) and anti-CD19 APC (eBioscience).NCR panel; anti-γδ PE; anti-CD8 Amcyan, anti-CD4 Percp-Cy5.5,anti-CD56 PE-Cy7, anti-CD3 APC-Cy7, anti-NKp46 Pacific Blue and anti-NKp30 APC (Biolegend,).All flow data unless specified was collected using the BD LSR II flow cytometer and analysed using BD FACSDiva ® software (BD Biosciences) (gating strategy in Supplementary Figure S2).For comparison of phenotyping expression data between SLL, B-CLL and HD, non-parametric Mann-Whitney testing was performed.All analysis was performed using Prism version 6.0, Graphpad software, San Diego, USA. ", "section_name": "Immunophenotyping", "section_num": null }, { "section_content": "NK cells were purified using EasySep™ Human NK cell enrichment kit (Stem Cell Technologies, Canada) (purity of sorted NK was around 90-95% of CD3 negative and CD56 positive lymphocytes) and activated overnight at 37 o C with Interferon-alpha (IFNα) (PeproTech, USA).For the K562 cytotoxicity assay, K562 cells were labeled with CFSE dye.The labeled K562 cells were then either incubated with RPMI (negative control) or in combination with activated NK cells at an E/T ratio of 0.5:1 on a 96 well plate for 16 hours.Cells were subsequently extracted and a fixed volume analyzed on the BD Accuri™ flow cytometer (BD Bioscience) to gain a relative cell count.% specific lysis was calculated by 100 × {1 -[(experimental group cell count)/(control cell count)]}.Propidium iodide dye was used for gating of the live and dead populations. For the NKG2D specific NK cytotoxicity assay, a 50:50 mixture of NKG2D ligand (ULBP6 (GenBank ID: AY039682.1)transfected Chinese hamster ovary (CHO) cell line labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and control transfected CHO cells labeled with 670 dye, were co-incubated with effector cells (IFN-α activated PBMCs) for 16hrs.After co-culture, the ratio of CFSE negative (670 dye positive, control CHO cells) to CFSE positive cells (ULBP6 transfected CHO cells) was calculated to determine the percentage of specific killing.% specific lysis was calculated by 100 × {1 -[(control ratio)/(experimental ratio)]}.The control ratios refer to the CFSE negative/CFSE positive without effectors cells, while the experimental ratio refers to the CFSE negative/CFSE positive with effectors cells. NKG2D blocking antibody (MAB139) was purchased from R&D system, and used at the concentration of 10 ug/ml in the blocking experiment. For the rituximab-induced NK cytotoxicity assay, target cells including primary B cells from HD and the B-CLL primary tumor cells were enriched using EasySep™ Human B cell enrichment kit (Stem Cell Technologies, Canada).The target cells were then either incubated with RPMI (negative control) or in combination with activated NK cells with or without Rituximab, at an E:T ratio of 5:1 on a 96 well plate for 16 hours.Cells were subsequently extracted and stained with anti-CD5-PE (Biolegend) and anti-CD19-FITC (Biolegend) to gate the B-CLL primary tumor cells and analysed on the BD Accuri™ flow cytometer to gain a relative cell count.The percentage of specific lysis was then calculated.Propidium iodide dye was used for gating of the live and dead populations. ", "section_name": "In vitro NK cell cytotoxicity", "section_num": null }, { "section_content": "The animal work was carried out under the Project license: 70/7793 and Personal License number: I031C5AD2.NOG (NOD/Shi-scid/IL-2Rg) mice were bred and maintained at the animal facility, University of Birmingham.The mice were treated for one week with baytril before tumor injection.At 8 weeks old, 12 NOG mice were irradiated with 1.25Gy.They were then injected subcutaneously with 1 × 10 7 K562 cells to the dorsal lateral thorax. At day 3, the mice were divided into 3 groups.Each group consisted of 4 mice and received either IL-2 only, IL-2 with 2 × 10 6 NK cells from HD or IL-2 with 2 × 10 6 NK cells from Patients with B-CLL.The NK cells were injected intravenously through the tail vein. The mice were culled seventeen days after receiving the K562 cells.The subcutaneous tumors were then removed and fixed in formaldehyde.After fixation, tissues were cut into sections of 5 µm with a cryomicrotome and then paraffin embedded (performed by Queen Elizabeth Hospital UK).The slides were then deparaffinised and rehydrated.Antigen retrieval was performed using a citrate and EDTA buffer.Slides were then washed with a blocking agent and incubated with primary antibodies.Four primary antibodies were used: anti-CD16, anti-CD56, anti-CD117 and anti-glycophorin C (Abcam, Cambridge, UK).After incubation, the slides were washed with PBS and monoclonal rabbit secondary antibodies were applied (Vector Laboratories, Burlingame CA, USA).The DAB solution (substrate buffer, DAB and chromogen) was then added before submerging in haematoxylin to counterstain nuclei.Images were obtained using a Nikon Eclipse 800 microscope.Tonsil controls were also used to optimize primary antibody concentrations and as a positive control. ", "section_name": "In-vivo NK cytotoxicity assay using xenograft NOD/SCID mouse model", "section_num": null }, { "section_content": "NK cells were first isolated from PBMCs using EasySep™ Human NK cell enrichment kit (STEMCELL biotechnology).The magnetically isolated NK cells were stained with anti-CD3 FITC, anti-CD56 Pe-Cy7 and LIVE/DEAD ® red stain for a further enrichment, whereby the CD3 negative and CD56 positive live cells were sorted with MoFlo™ cell sorter (Beckman Coulter).The purity of all the sorted NK population was around 99% of CD3 negative and CD56 positive lymphocytes.The sorted NK cell populations were sent to AROS Applied Biotechnology A/S (Aarhus N, Denmark) as dry cell pellets.Total RNA was extracted, labelled and hybridized to GeneChip ® Human Transcriptome Array 2.0 (Affymetrix, USA).Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-4403.Raw data was processed using Affymetrix's Expression Console software using default RMA parameters.Statistical analysis of expression data and heatmap generation was performed using d-CHIP software (http://www.dchip.org).The T test in d-CHIP is Welch modified two-sample t-test.Multiple comparison adjustment was carried out to include permutation to make sure the median FDR (False Discovery Rate) was < 5%.Venn diagrams were constructed from up or downregulated gene expression in B-CLL or Patients with SLL compared to HD. ", "section_name": "NK cell sorting and microarray gene transcription profiling", "section_num": null } ]	[ { "section_content": "The authors would like to thank the funding from The Wellcome Trust and Bloodwise. There are no conflicts of interest to declare. ", "section_name": "Footnotes", "section_num": null }, { "section_content": "The authors would like to thank the funding from The Wellcome Trust and Bloodwise. ", "section_name": "Footnotes", "section_num": null }, { "section_content": "There are no conflicts of interest to declare. ", "section_name": "CONFLICTS OF INTEREST", "section_num": null } ]
10.1590/s1516-84842010000400012	Emprego da citometria de fluxo na avaliação do perfil imunofenotípico de pacientes com leucemia linfocítica crônica	A leucemia linfocitica cronica (LLC-B) e uma proliferacao clonal de linfocitos B maduros caracterizada por curso clinico indolente. Biologicamente esta clonalidade e caracterizada pela baixa expressao de imunoglobulina de superficie (sIg) com restricao a uma unica cadeia leve de imunoglobulina, associada a alta expressao do antigeno CD5 e positividade a antigenos relacionados a linfocitos B tais como: CD19, CD20 e CD23 e negatividade ao FMC7. O perfil imunologico e a analise morfologica das celulas linfoides sao os principais meios para o diagnostico diferencial LLC-B de outras doencas linfoproliferativas cronicas. O objetivo deste estudo foi avaliar o padrao de expressao de uma variedade de antigenos de membrana em celulas leucemicas procedentes de pacientes com LLC-B. No presente estudo, amostras de sangue periferico de 80 pacientes com LLC-B foram analisados por citometria de fluxo multiparametrica juntamente com analises hematologicas de rotina, com um painel de anticorpos monoclonais (AcMo): CD45/CD14, CD3/CD19/CD45, CD4/CD8/CD3, CD20/CD5/CD3, CD3/CD16-56/CD45, CD2/CD7, FMC7/CD23, CD103/CD22/CD20, HLADR/CD38, CD10/CD19, CD1a, CD11b alem de IgM/gD, e cadeias leves das imunoglobulinas kappa e lambda visando a deteccao sIg e do perfil de restricao clonal das cadeia leves das imunoglobulinas. Os dados hematologicos foram obtidos a partir do analisador hematologico e as analises citomorfologicas em distensao sanguinea coradas pelo Leishmann. As amostras deste estudo foram procedentes de 45 homens e 35 mulheres, com idade variando entre 55 e 84 anos (media de 65 anos). O hemograma revelou contagem total de celulas branca variando de 10,0-42,0 x 109/l. (media de 50,0 x 109/l) e contagem de linfocitos superior a 5,0 x 109/l em todos os casos. As celulas neoplasicas demonstraram um fenotipo caracteristico para LLC-B (CD5+/CD19+/CD20+/HLADR+/CD23+) na maioria dos casos, associados a ausencia de expressao para marcadores de celulas T (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 e FMC7. As celulas leucemicas da maioria dos pacientes expressaram tambem IgM e IgD de baixa intensidade com predominio da restricao da cadeia leve kappa, na maioria dos casos (59,7%). A presente observacao destaca a importância da imunofenotipagem para o correto diagnostico das sindromes linfoproliferativas cronicas e sendo o painel de AcMo utilizado capaz de estabelecer a confirmacao diagnostica da B-CLL	[ { "section_content": "B (CD5+/CD19+/CD20+/HLADR+/CD23+) na maioria dos casos, associados à ausência de expressão para marcadores de células T (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 e FMC7.As células leucêmicas da maioria dos pacientes expressaram também IgM e IgD de baixa intensidade com restrição clonal da cadeia leve kappa, na maioria dos casos (59,7%).A presente observação destaca a importância do imunofenotipagem para correto diagnóstico das síndromes linfoproliferativas crônicas e sendo o painel de AcMo utilizado capaz de estabelecer a confirmação diagnóstica da LLC-B. Palavras-chave: Leucemia linfocítica crônica; citometria de fluxo; imunofenotipagem. ", "section_name": "", "section_num": "" }, { "section_content": "Chronic lymphocytic leukemia (B-CLL) is a clonal proliferation of mature B lymphocytes characterized by indolent clinical course.Biologically this clonallity is characterized by low expression of surface immunoglobulin (sIg) with restriction to a single immunoglobulin light chain associated with high expression of CD5 antigen and positivity to B cell antigens lymphocytes such as CD19, CD20 and CD23 and negativity to FMC7.The immunological profile and morphological analysis of lymphoid cells are the main means for the differential diagnosis of B-CLL from other chronic lymphoproliferative diseases.The aim of this study was to evaluate the expression pattern of a variety of membrane antigens in leukemic cells originating from patients with B-CLL.In this study, peripheral blood samples from 80 patients with B-CLL were analyzed by multiparametric flow cytometry in addition to routine hematologic exams, using a panel of monoclonal antibodies (MoAb): CD45/CD14, CD3/CD19/CD45, CD4/CD8/CD3, CD20/CD5/CD3, CD3/CD16-56/CD45, CD2/ CD7, FMC7/CD23, CD103/CD22/CD20, HLADR/CD38, CD10/ CD19, CD1a, CD11b and also IgM/gD, kappa and lambda immunoglobulin light chains for the detection of surface Avaliação: A RBHH publica os resumos e abstracts de teses da área apresentados em entidades que tenham programas de pós-graduação reconhecidos pelo MEC/Capes e considera a obtenção do título suficiente para sua publicação na forma como se propõe a seção.Suporte Financeiro: 1-Fapern (Fundação de Apoio a Pesquisa do Estado do Rio Grande do Norte); 2-Hemocentro Dalton Barbosa Cunha -Hemonorte; 3-Pro-Reitoria de Pesquisa da UFRN-Propesq. Recebido: 6/3/2010 Aceito após modificações: 7/5/2010 Rev. Bras. Hematol. Hemoter. 2010;32(4):337-338 Resumo de Tese immunoglobulin and clonal restriction for immunoglobulin light chain.The Hematological data were obtained from the hematological analyzer and cytomorphological analysis in blood film stained by Leishmann.The study samples consisted of 45 men and 35 women, ages ranging from 55 to 84 years (mean 65 years).Complete white blood count showed count ranging from 10.0 to 42.0 x 10 9 /L.(mean 50.0 x 10 9 /L) and lymphocytes count greater than 5.0 x 10 9 /l in all cases.The neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/CD20+/HLADR+/CD23+) in the vast majority of the cases, associated to failed to stain for T cell markers (CD1a, CD2, CD4, CD3, CD7, CD8), CD103, CD14 and FMC7.Leukemic cells of most patients also expressed low intensity of IgM and IgD with clonal restricted kappa light chain in most cases (59,7%).This observation highlights the importance of immunophenotyping for correct diagnosis of chronic lymphoproliferative syndromes and the panel of MoAb used was sufficient for diagnostic confirmation of B-CLL. Key words: Chronic lymphocytic leukemia; flow cytometry; immunophenotyping. ", "section_name": "Abstract", "section_num": null } ]	[]
10.1038/s41467-018-04283-9	NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence	<jats:title>Abstract</jats:title><jats:p>Senescent cells interact with the surrounding microenvironment achieving diverse functional outcomes. We have recently identified that NOTCH1 can drive ‘lateral induction’ of a unique senescence phenotype in adjacent cells by specifically upregulating the NOTCH ligand JAG1. Here we show that NOTCH signalling can modulate chromatin structure autonomously and non-autonomously. In addition to senescence-associated heterochromatic foci (SAHF), oncogenic RAS-induced senescent (RIS) cells exhibit a massive increase in chromatin accessibility. NOTCH signalling suppresses SAHF and increased chromatin accessibility in this context. Strikingly, NOTCH-induced senescent cells, or cancer cells with high JAG1 expression, drive similar chromatin architectural changes in adjacent cells through cell–cell contact. Mechanistically, we show that NOTCH signalling represses the chromatin architectural protein HMGA1, an association found in multiple human cancers. Thus, HMGA1 is involved not only in SAHFs but also in RIS-driven chromatin accessibility. In conclusion, this study identifies that the JAG1–NOTCH–HMGA1 axis mediates the juxtacrine regulation of chromatin architecture.</jats:p>	[ { "section_content": "ellular senescence is an autonomous tumour-suppressor mechanism that can be triggered by pathophysiological stimuli including replicative exhaustion, exposure to chemotherapeutic drugs and hyper-activation of oncogenes, such as RAS 1 .Persistent cell cycle arrest is accompanied by diverse transcriptional, biochemical and morphological alterations.These senescence hallmarks include increased expression and secretion of soluble factors (senescence-associated secretory phenotype (SASP)) 2,3 and dramatic alterations to chromatin structure 1,4,5 .Importantly, the combination, quantity and quality of these features can vary depending on the type of senescence.Senescent cells have profound non-cell autonomous functionality.The SASP can have either protumorigenic or antitumorigenic effects and act in an autocrine or paracrine fashion 2,[6][7][8] .In addition, we have recently identified that NOTCH signalling can drive a cellcontact-dependent juxtacrine senescence 9 . The NOTCH signalling pathway is involved in a wide array of developmental and (patho-)physiological processes.NOTCH has roles in differentiation and stem cell fate 10 and perturbations have been linked to tumorigenesis where NOTCH can have either oncogenic or tumour-suppressive functionality 11 .The pathway involves proteolytic cleavage of the NOTCH receptor upon contact-mediated activation by a ligand of the JAGGED (JAG) or DELTA family on the surface of an adjacent cell.The cleaved NOTCH-intracellular domain translocates to the nucleus where, together with transcriptional co-activators such as mastermindlike 1 (MAML1), it drives transcription of canonical target genes, including the HES and HEY family of transcription factors 10 .NOTCH signalling has also been shown to induce a type of senescence, NOTCH-induced senescence (NIS), where cells are characterised by distinct SASP components 9,12 .Recently, we showed that during NIS there is a dramatic and specific upregulation of JAG1 that can activate NOTCH1 signalling and drive NIS in adjacent cells ('lateral induction') 9 . During senescence, particularly in oncogenic RAS-induced senescent (RIS) fibroblasts, characteristic changes to chromatin culminate in the formation of senescence-associated heterochromatic foci (SAHFs) 13 rearrangement of existing heterochromatin 14 .Other alterations include the formation of senescence-associated distention of satellites (SADS) 15 .SAHF formation is dependent on chromatin-bound highmobility group A (HMGA) proteins, particularly HMGA1 16 .These are a family of architectural proteins, consisting of HMGA1 and HMGA2, which bind to the minor groove of ATrich DNA via three AT-hook domains to alter chromatin structure 17,18 .Despite a critical role in the formation of SAHFs during senescence, HMGA proteins are also important during development where they promote tissue growth 19,20 and regulate differentiation [21][22][23][24] .Furthermore, many studies have demonstrated an association between high HMGA1 expression and aggressive tumour biology 25,26 . Chromatin accessibility at regulatory elements including promoters and enhancers is highly correlated with biological activity 27 .High-throughput sequencing using FAIRE-seq, a method that identifies open and closed chromatin based on phenol separation 28 , has revealed that, in cells that have undergone replicative senescence, previously heterochromatic domains enriched for various repeat elements become more accessible while euchromatic domains undergo condensation 29 .However, it remains unknown how chromatin accessibility is altered in RIS and NIS cells. Here we characterise the chromatin phenotype in RIS and NIS cells.We demonstrate that these two types of senescent cells exhibit distinct chromatin structures at microscopic and nucleosome scales. Both gain multiple chromatin accessible regions, which are often exclusive between RIS and NIS.Strikingly, we find that autonomous and non-cell autonomous activation of the NOTCH signalling pathway in RIS cells can repress SAHFs and the formation of RISdriven chromatin-accessible regions, partially by transcriptional repression of HMGA1.Our study demonstrates that chromatin structure and the nucleosome landscape can be regulated through juxtacrine signalling.The relationship between these two prominent tumour-associated genes, HMGA1 and NOTCH1, may also have prognostic value in vivo. ", "section_name": "C", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "SAHFs.We have previously demonstrated that ectopic NOTCH1-intracellular domain (N1ICD), an active form of NOTCH1 (Fig. 1a), can drive NIS that is distinct from RIS in terms of SASP composition 9 and noticed that NIS cells also have a unique chromatin structure. To examine the relationship between NOTCH1 and chromatin structure, we introduced ectopic N1ICD into IMR90 human diploid fibroblasts (HDFs) stably expressing a 4-hydroxytamoxifen (4OHT)-inducible oestrogen receptor-oncogenic HRAS fusion protein (IMR90 ER:HRAS G12V cells) 30 .Ectopic expression of N1ICD alone induced senescence with dramatically enlarged nuclei, even larger than in RIS (Fig. 1b,c).Similarly to RIS, NIS cells formed SADS, a more common chromatin feature of senescence than SAHFs (Supplementary Fig. 1a) 15 .However, in marked contrast to RIS, NIS cells lacked SAHFs (Fig. 1b,d). To ask whether NIS cells simply lack SAHFs or whether N1ICD actively modulates chromatin structure, we expressed N1ICD in the presence of HRAS G12V induced using 100 nM of 4OHT for 6 days.Interestingly, N1ICD in the context of RIS also resulted in a dramatic enlargement of nuclei but a complete ablation of SAHF formation (Fig. 1b-d).This was emphasised by a 'smoothening' of chromatin as indicated by a marked reduction in the standard deviation of 4,6-diamidino-2-phenylindole (DAPI) signal measured within individual nuclei (Fig. 1b,c; Supplementary Fig. 1b-d), We have previously shown that ectopic N1ICD in the RIS context results in senescence with SASP composition broadly similar to NIS 9 .Thus our data indicate that NOTCH is dominant over RIS in terms of chromatin phenotype as well as SASP composition. In IMR90 ER:HRAS G12V cells, RIS develops progressively over a time period of ~6 days following the addition of 4OHT 30 .NOTCH1 signalling is temporally regulated during RIS, where cleaved and active N1ICD is transiently upregulated before downregulation at full senescence 9 .To examine the temporal effects of NOTCH1 signalling on SAHF formation, we performed a time course experiment in IMR90 ER:HRAS G12V cells.Cells were retrovirally infected with a dominant-negative form of MAML1 fused to mVenus (dnMAML1-mVenus) or treated with the γ-secretase inhibitor N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) to repress downstream signalling by N1ICD (Fig. 1a).We found that a greater number of SAHF-positive cells were formed and that these accumulated at earlier time points when NOTCH1 signalling was repressed (Fig. 1e).Furthermore, a dose-dependent effect was evident where higher concentrations of DAPT resulted in a greater proportion of cells developing SAHF during RIS (Supplementary Fig. 1e).SAHFs are not typically prominent in DNA damage-induced senescence (DDIS) in IMR90 cells 1 .However, DAPT significantly promoted SAHF formation in DDIS (by etoposide) (Supplementary Fig. 1f).To determine whether NOTCH1 activity can reverse SAHF after they have formed, we infected IMR90 cells with doxycycline (DOX)inducible N1ICD-FLAG and constitutive HRAS G12V .The addition of DOX after the establishment of senescence was sufficient to reduce the number of SAHF-positive cells and the standard deviation of DAPI signal, suggesting some degree of reversibility (Supplementary Fig. 1g).Together, our data suggest that NOTCH signalling has a chromatin 'smoothening' effect that antagonises SAHF formation. Non-cell autonomous regulation of SAHFs.N1ICD-expressing cells can induce NIS in adjacent normal cells, at least in the case of IMR90 fibroblasts 9 .To determine whether N1ICD-expressing cells can also alter chromatin structure in adjacent cells, we performed co-cultures between mRFP1-expressing IMR90 ER: HRAS G12V and IMR90 cells expressing DOX-inducible N1ICD-FLAG in the presence and absence of 4OHT and DOX (Fig. 2a).Strikingly, co-culture with N1ICD-expressing IMR90 cells was sufficient to repress SAHF formation in adjacent RIS (red) cells (Fig. 2b,c). Of the canonical NOTCH1 ligands, we have previously observed a strong and unique upregulation of JAG1 following ectopic N1ICD expression, which we found to be responsible for the juxtacrine transmission of NIS 9 .We reasoned that N1ICDmediated upregulation of JAG1 and subsequent 'lateral induction' of NOTCH1 signalling is a likely mechanism by which SAHFs are regulated non-autonomously.To test this hypothesis, we expressed ectopic JAG1 fused to mVenus (JAG1-mVenus) in retinal pigment epithelial (RPE1) cells.We confirmed cell surface expression of ectopic JAG1 by flow cytometry (Supplementary Fig. 2a) before co-culturing with mRFP1-expressing IMR90 ER: HRAS G12V cells.RPE1 JAG1-mVenus cells, but not control RPE1 cells, significantly repressed the formation of SAHFs (Fig. 2e,f).Note that this repression did not occur when these two types of cells were co-cultured without physical contact in a transwell format (Supplementary Fig. 2b).Our data suggest a mechanism by which lateral induction of NOTCH signalling by JAG1 can block SAHFs in the context of RIS; i.e. higherorder chromatin structure can be regulated through cell-cell contact. ", "section_name": "NOTCH1 reprogrammes chromatin structure and abrogates", "section_num": null }, { "section_content": "To unravel the mechanisms underpinning NOTCH1-dependent repression of SAHFs, we re-analysed previously published RNAseq data generated from IMR90 cells expressing HRAS G12V and N1ICD 9 .We found that N1ICD dramatically represses the expression of HMGA1 and HMGA2 (Supplementary Fig. 3a), critical components of SAHF structure 16 . To validate that NOTCH1 signalling represses HMGAs, we introduced constitutive N1ICD into IMR90 ER:HRAS G12V cells.Ectopic N1ICD significantly repressed HMGA1 and HMGA2 at an mRNA and protein level in both the presence and absence of 4OHT-induced HRAS G12V (Fig. 3a,b).The enforced expression of N1ICD after senescence establishment also resulted in the reduction of HMGA1 albeit to a lesser extent than pre-senescence N1ICD expression (Supplementary Fig. 1g).N1ICD has a similar effect on HMGA1 and 2 protein levels when expressed in other cell lines in the absence of HRAS G12V , suggesting a conserved mechanism (Supplementary Fig. 3b).In the DOX-inducible N1ICD-FLAG system, inhibition of NOTCH1 signalling by coexpression of dnMAML1-mVenus was sufficient to rescue N1ICD-mediated repression of HMGA1 and HMGA2 (Fig. 3c,d), suggesting the effect is dependent on the canonical pathway of NOTCH signalling. Finally, we used IMR90 ER:HRAS G12V cells expressing DOXinducible N1ICD-FLAG to investigate whether ectopic reexpression of EGFP-tagged HMGA1 is sufficient to rescue SAHFs.The introduction of EGFP-HMGA1 resulted in a partial, but significant, rescue of SAHF-positive cells when cells were treated with DOX and 4OHT (Fig. 3e). Collectively, our data suggest that NOTCH1 signalling represses the formation of SAHFs at least partially by inhibiting HMGAs. Non-cell autonomous inhibition of HMGAs.To determine whether HMGAs are repressed non-autonomously by JAG1 expressing cells, we performed further co-cultures between RPE1 cells retrovirally infected with JAG1-mVenus and IMR90 cells ectopically expressing a cell surface marker, rat-Thy1, allowing for subsequent isolation using magnetic-activated cell sorting (MACS) (Fig. 3f).As expected, IMR90 cells co-cultured with JAG1-expressing cells upregulated canonical NOTCH1 target genes, HEY1 and HEYL.Both HMGA1 and HMGA2 were significantly repressed in the same IMR90 cells (Fig. 3f), demonstrating that HMGA proteins can be repressed non-cell autonomously. Altered chromatin accessibility in RIS and NIS.To investigate whether NOTCH1 influences chromatin structure at a higher resolution, we employed ATAC-seq (assay for transposaseaccessible chromatin using sequencing) 31 .This method exploits a hyperactive Tn5 transposase that inserts sequencing adapters into regions of accessible chromatin.Following adapter-primed PCR amplification, these regions were sequenced to identify accessible regions of chromatin genome wide (Fig. 4a). We generated at least three replicates from IMR90 ER: HRAS G12V cells expressing N1ICD-FLAG or a control vector and induced with 4OHT or not.For simplicity, these conditions were labelled as 'Growing', 'RIS', 'NIS' and 'N+RIS' (expressing both N1ICD and RAS).Using a previously published normalisation approach 32 , we generated normalised coverage files that appeared comparable to each other, especially around housekeeping genes (Fig. 4b).Most of the samples, excluding a single replicate from the NIS and N+RIS conditions (which were excluded from downstream analysis), were of high quality with a 'reads in peaks' percentage (RiP%) of >10% (Supplementary Fig. 4a).Replicates clustered well by unbiased principal component analysis (PCA) (Supplementary Fig. 4b).Moreover, our samples clustered with publically available ATAC-seq and DNase-seq data generated from IMR90 cells (Supplementary Fig. 4c), but separated from other cell types (BJ, HaCaT, MCF710A and HEKn cells). Using MACS peak calling, we found that the number of peaks identified in each replicate of a condition was similar and that, in general, chromatin accessibility was dramatically increased in RIS (145,649 consensus peaks detected in ≥2 replicates) and NIS cells (149,877 peaks) relative to growing cells (83,920 peaks) (Supplementary Fig. 5a).To quantitatively identify regions of altered accessibility in RIS and NIS cells relative to growing cells, we performed differential binding analysis using both edgeR 33,34 and THOR 35 before taking only regions identified by both methods for downstream analysis (Supplementary Data 1).Using this stringent approach, we identified 44,556 regions that become significantly more accessible (opened) and 9603 regions that become significantly less accessible (closed) in RIS cells relative to growing cells.In NIS cells, 20,499 regions became more accessible and 15,444 regions less accessible (Fig. 4c).Despite the robust gain of chromatin accessibility in both types of senescence, there were relatively few shared sites (Fig. 4d). A previous study mapping chromatin accessibility in replicatively senescent cells using FAIRE-Seq found that gene-distal regions, especially repeat regions, become relatively more open whereas genic regions become closed compared to growing fibroblasts 29 .Consistently, regions of increased accessibility in RIS and NIS cells were enriched at gene-distal sites (Supplementary Fig. 5b) with the majority of opened regions mapping to enhancer, intergenic, intronic and repeat regions (Fig. 4e).Many of these repeat regions were further annotated as long interspersed elements, long-terminal repeats, short interspersed elements and simple repeat regions (Supplementary Fig. 5c,d), although these values may be underestimated due to the exclusion of multi-mapping reads from our data.Many of the regions that became less accessible in RIS cells relative to growing cells were closer to transcriptional start sites (TSSs) (Supplementary Fig. 5b) and mapped to exons, CpG-islands and untranslated regions (UTRs) (Fig. 4e).In contrast to replicative senescence 29 gene-distal elements (Supplementary Fig. 5b, Fig. 4e).Therefore, while RIS largely mirrors replicative senescence, NIS is characterised by remodelling (both opening and closing) of genedistal regions. Altered accessibility of genes reflects expression.Chromatin accessibility at regulatory elements has been correlated with gene expression 27 .To determine whether genic alterations to chromatin accessibility in RIS and NIS reflects gene expression, we assigned regions of altered accessibility (opened or closed in RIS or NIS) to genes if within 500 bp of a TSS (Fig. 4f).On average, genes that were opened in RIS relative to growing cells were also transcriptionally upregulated by mRNA-seq in RIS relative to growing cells (Fig. 4f, top).Genes that were opened in NIS cells were transcriptionally upregulated in NIS cells, while less accessible genes were transcriptionally repressed (Fig. 4f, bottom).Consistent with our previous RNA-seq data 9 , genes that became more accessible in RIS were significantly enriched within gene ontology (GO) terms such as 'inflammatory response' and 'cytokine secretion', reflecting the inflammatory secretome produced by RIS cells (Fig. 4g).Genes that became less accessible in RIS were enriched within GO terms such as 'regulation of cell cycle' (Fig. 4g), perhaps reflecting non-proliferative features of RIS (although average gene expression of this gene set was not significantly altered).Unbiased motif enrichment analysis revealed that regions opened in RIS were highly enriched for the C/EBPβ-binding motif (Supplementary Fig. 5e), consistent with the important role of C/EBPβ in regulating the inflammatory SASP 3 .Regions opened in NIS were enriched with the RBP-Jbinding motif (Supplementary Fig. 5 f), a critical DNA-binding factor downstream of NOTCH signalling 11 .Normalised ATACseq coverage files, when viewed using a genome browser (Fig. 4b, Supplementary Fig. 6a-d), demonstrated increased accessibility around transcriptionally activated genes.We also noted that, while the accessibility at many promoters was unaltered in RIS cells, some transcriptionally activated genes, such as IL1A and HMGA1, were proximal to enhancer elements that became more accessible (Fig. 4b, Supplementary Fig. 6a).Together, these data demonstrate that RIS and NIS cells have unique open chromatin landscapes and that (gene proximal) alterations reflect their transcriptional landscapes. NOTCH signalling antagonises chromatin opening in RIS.By unbiased clustering of ATAC-seq data, we observed a greater correlation between NIS and N+RIS cells than between RIS and N+RIS cells (Fig. 5a).This suggests a dominant effect of N1ICD over RAS on the nucleosome scale, consistent with our previous observations for SASP components 9 and SAHFs (Fig. 1d). To determine whether NOTCH1 signalling can repress the chromatin alterations observed in RIS in favour of a 'NIS-like' chromatin landscape, we focussed on the 44,556 regions that became significantly more accessible in RIS cells relative to growing cells (referred to as 'RIS-driven accessible regions', Fig. 4c) and the 20,499 regions that became significantly more accessible in NIS cells relative to growing cells (referred to as 'NIS-driven accessible regions', Fig. 4c).By comparing chromatin accessibility of N+RIS cells with RIS cells we found that formation of many RIS-driven accessible regions (62.7%) were repressed by N1ICD expression (Fig. 5b).N1ICD expression also increased the accessibility of NIS-driven accessible regions (Fig. 5b).When viewed in the genome browser, it was evident that N1ICD expression can repress the formation of accessible regions located at enhancer elements upstream of the HMGA1 promoter in RIS cells (Supplementary Fig. 6a), although we failed to detect any alterations at the HMGA2 locus (Supplementary Fig. 6b), providing a potential mechanism for NOTCH1mediated repression of HMGA1. HMGA proteins have previously been shown to affect chromatin compaction.To determine whether repression of HMGA1 is a mechanism by which N1ICD can repress formation of RIS-driven accessible regions, we generated additional ATACseq samples from IMR90 ER:HRAS G12V cells expressing a short hairpin against HMGA1 16 and treated with 4OHT, hereafter referred to as 'RIS+shHMGA1'.By comparing RIS+shHMGA1 with RIS, we identified 8909 RIS-driven accessible regions that were dependent on HMGA1 (Fig. 5c).Of these, 69.9% (6168) were also repressed by N1ICD (Fig. 5d).These analyses illustrate that a subset of RIS-driven accessible regions can be repressed by N1ICD, possibly by HMGA downregulation.However, HMGA1 knockdown was not sufficient to induce the formation of NIS-driven accessible regions (Fig. 5c), suggesting an HMGA1-independent mechanism in the formation of these sites.RIS-driven accessible regions (opened in RIS) were significantly more AT-rich than NISdriven accessible regions (opened in NIS) or regions with reduced accessibility (Fig. 5e), supporting the involvement of HMGA1 in the formation of RIS-driven accessible regions. To validate the above approach, we used our normalised coverage files to perform unbiased k-means clustering centred around accessible regions that were altered in either RIS or NIS cells (opened or closed relative to growing cells) (Fig. 5f).Accessible regions separated into clusters that were dominated by either the RIS (clusters 1 and 2) or NIS (clusters 3 and 4) conditions.Strikingly, the signal in RIS-dominated clusters, cluster 2 in particular, was reduced in the N+RIS and RIS +shHMGA1 conditions when compared to the RIS condition (Fig. 5f).Consistently, cluster 2 was more AT-rich than cluster 1 (Fig. 5g), supporting a role for HMGA1.Notably, while peaks in clusters 3 and 4 were increased in the N+RIS condition, they did not increase in the RIS+shHMGA1 condition, reinforcing an HMGA1-independent mechanism of chromatin opening in NIS (Fig. 5f).Therefore, in line with microscopic SAHF structures, N1ICD alters chromatin structure in RIS at the nucleosome scale in part by repressing HMGA1 expression. .n = 3 biological replicates except for Hep3B cultures where n = 6.c SAHF quantification in IMR90 ER:HRAS G12V cells expressing mRFP+100 nM 4OHT±10 µM DAPT.d Schematic showing experimental set-up.IMR90 ER:HRAS G12V cells expressing mRFP were cultured with tumour cell lines +100 nM 4OHT before flow sorting to isolate red cells.e qRT-PCR of mRNA isolated from the cells described in c. n = 3 biological replicates.f Immunoblotting of JAG1 in the cells indicated.g Quantification of SAHFs in IMR90 ER:HRAS G12V expressing mRFP co-cultured with the tumour cells indicated for 6 days +100 nM 4OHT.n = 3 biological replicates.h Volcano plots showing regions of altered accessibility in RIS cells co-cultured with MCF7, A549 and Hep3B cells (as in c) relative to RIS cells cultured alone.Regions that also become more accessible in RIS (red) and NIS (purple) vs. growing are indicated where numbers indicate the total number of significant alterations (log 2 fold change <-0.58 or >0.58 and FDR < 0.01) and <-1 indicates the number with a log 2 fold change of <-1.i Number of more accessible regions in RIS (identified in Fig. 4c) that are repressed by co-culture with MCF7, A549 and Hep3B.b, c, e, g Statistical significance calculated using one-way ANOVA with Tukey's correction for multiple comparisons; p ≤ 0.05, p ≤ 0.01, **p ≤ 0.001, NS = not significant Non-cell autonomous regulation of SAHFs by tumour cells.Both HMGA1 and NOTCH1 can act as oncogenes or tumour suppressors in a context-dependent manner.We reasoned that the relationship between these two genes might also be important in the tumour microenvironment and asked whether tumour cells expressing JAG1 can affect HMGA1 expression and chromatin structure in adjacent fibroblasts. To answer this question, we used the Cancer Cell Line Encyclopedia 36 to identify tumour cell lines that express low (MCF7), medium (A549) and high (Hep3B) levels of JAG1, which we confirmed by immunoblotting (Fig. 6a).Co-culture of tumour cell lines with IMR90 cells expressing both ER:HRAS G12V and mRFP1 in the presence of 4OHT was sufficient to repress SAHF formation in red (RIS) cells in a contact-dependent manner (Fig. 6b; Supplementary Fig. 7a).The number of SAHF-positive red cells inversely correlated with the level of JAG1 expressed by the tumour cell lines (Fig. 6b).Non-autonomous inhibition of SAHF formation in the co-culture system was completely abrogated by DAPT, suggesting the effect is dependent on the canonical NOTCH pathway (Fig. 6b,c).Consistent with our previous experiments (Fig. 1e, Supplementary Fig. 1e), the addition of DAPT was sufficient to increase the percentage of SAHF-positive IMR90 cells above basal levels both in monoculture (Fig. 6c) and co-culture (Fig. 6b). To determine whether tumour cell lines can induce NOTCH1 signalling and repress HMGAs non-autonomously, we repeated the co-cultures and isolated the IMR90 ER: HRAS G12V mRFP1 cells using flow cytometry (Fig. 6d, Supplementary Fig. 7b).We found a dramatic upregulation of the canonical NOTCH1 target gene HEYL and a concurrent downregulation of HMGA1 and HMGA2 in fibroblasts cocultured with JAG1-expressing tumour cells, particularly A549 and Hep3B cells (Fig. 6e).Two other canonical target genes, HES1 and HEY1, were not dramatically upregulated by JAG1expressing cell lines (Supplementary Fig. 7c).Although HEYL, HEY1 and HES1 are known as 'canonical targets' of NOTCH, their transcriptional regulation by NOTCH signalling is highly complex: for example, unique combinations of, or interactions between, NOTCH ligands and receptors can provide preferential induction of certain targets 37,38 .Different tumour cell lines might differentially express other NOTCH ligands (in addition to JAG1) or other NOTCH pathway modulators, conferring additional complexity. To further determine whether the effect described above is JAG1-dependent, we used CRISPR-Cas9 technology to generate A549 cells with bi-allelic knockout of endogenous JAG1.Two knockout clones were isolated; clone 1 had a 5 bp deletion in the first allele and a 1 bp deletion in the second allele while clone 2 had a 1 bp deletion in the first allele and a 14 bp deletion in the second allele (Supplementary Fig. 7d).A control clone was generated by transfecting cells with Cas9 but omitting guide RNA (-gRNA control cells).We found that both clones 1 and 2 had reduced JAG1 levels by immunoblotting (Fig. 6f) and cell-surface JAG1 (plus JAG2) levels by flow cytometry (Supplementary Fig. 7e).Proliferation was not substantially altered in JAG1knockout cells relative to control or parental cells (Supplementary 7f).In contrast to control or parental A549 cells, co-culture of JAG1-knockout A549 cells with red IMR90 ER:HRAS G12V cells in the presence of 4OHT had little effect on SAHF formation in red (RIS) cells (Fig. 6g). In addition to JAG1-knockout A549 cells, we generated MCF7 cells containing DOX-inducible JAG1 fused to mVenus (JAG1-mVenus).By immunoblotting, we observed low-level expression of JAG1-mVenus even in the absence of DOX, likely caused by 'leaky' transcription (Supplementary Fig. 7g).Addition of 10 ng/ mL of DOX to the culture was sufficient to induce JAG1 to comparable levels as those observed endogenously in Hep3B cells (Supplementary Fig. 7g).Co-culture of MCF7 cells containing inducible JAG1 with red IMR90 ER:HRAS G12V cells was sufficient to reduce the number of SAHF-positive red cells even in the absence of DOX (reflecting the slightly increased levels of JAG1) and completely repress SAHF formation in red cells in the presence of DOX (Supplementary Fig. 7h).While we cannot exclude the effects of other cell-contact-mediated signalling pathways on chromatin structure, our data together demonstrate that JAG1-expressing tumour cells can repress SAHF formation in adjacent senescent cells in a JAG1-dependent manner. Non-cell autonomous regulation of chromatin accessibility.Next, we asked whether tumour cell lines could repress the formation of RIS-driven accessible regions in fibroblasts, as was the case for ectopic N1ICD (Fig. 5b).Utilising flow cytometry, we isolated 4OHT-induced IMR90 ER:HRAS G12V mRFP1 cells after co-culture with tumour cell lines and performed ATAC-seq (Fig. 6d).We found that 66% (29,507), 72% (32,364) and 75% (33,581) of RIS-driven accessible regions were significantly repressed by co-culture with MCF7, A549 and Hep3B cells, respectively (Fig. 6h, i; Supplementary Fig. 8a,b).Co-culture with MCF7, A549 or Hep3B cells induced opening of 6948, 10,303 and 14,064 NIS-driven accessible regions, respectively (Fig. 6h).These data correlated well with the ability of the tumour cell lines to repress SAHFs in adjacent IMR90 (Fig. 6b) and the JAG1 levels expressed by each line (Fig. 6a).RIS-driven accessible regions repressed by co-culture with tumour cell lines overlapped well with each other and with regions repressed by ectopic N1ICD (Supplementary Fig. 8c,d).These data suggest that tumour cells expressing JAG1 can dramatically alter the chromatin landscape of adjacent stromal cells at the nucleosome level. HEYL and HMGA1 anti-correlate in multiple tumour types.If NOTCH1 signalling inhibits HMGA1 in vivo, we would expect an anti-correlation between NOTCH1 activity and HMGA1 expression in human tumour samples.To test this, we first performed a pan-tissue-type analysis using expression microarray data from the R2 database (http://r2.amc.nl) by comparing the expression of HMGA1 and canonical NOTCH1 target genes.When Z-score expression values were analysed in 36,846 human samples, we observed a significant negative correlation between HMGA1 and HEYL (R = -0.356,p < 0.0001) and HMGA1 and HEY1 (R = -0.281,p < 0.0001), but no correlation between HMGA1 and HES1 (Supplementary Fig. 9a,b,c).Interestingly, HEYL and HEY1, but not HES1, were also significantly upregulated in IMR90 fibroblasts co-cultured with JAG1-expressing RPE1 cells (Fig. 3g).To study the prognostic importance of this relationship, we used the web-based tool KM-plotter 39,40 and found that patients with low HMGA1 or high HEYL have a significantly better prognosis in lung adenocarcinoma, but not in lung squamous cell carcinoma (SCC) (Supplementary Fig. 9d,e), suggesting that the relationship between these proteins may have prognostic value in certain types of cancer.High HEY1 levels were prognostic of better overall survival in both types of lung cancer patient (Supplementary Fig. 9d,e). As microarray data can be dependent on the quality of the probe used, we analysed the co-expression of HMGA1 and HEYL or HEY1 using RNA-seq data generated by The Cancer Genome Atlas (TCGA) Research Network 41 (http://cancergenome.nih.gov).There was a significant negative correlation between HMGA1 and HEYL in the majority of tumour types analysed (Fig. 7a) and a particularly strong anti-correlation in lung SCC (Fig. 7b) (R = -0.4842;p = < 0.0001).When TCGA patients with lung SCC were categorised based on expression into 'HMGA1 high-HEYL low' and 'HMGA1 low-HEYL high' tumours, patients in the former category had a better overall survival (Fig. 7c) (p = 0.00316).We also found a significant negative correlation between HMGA1 and HEY1 in various cancer types, which were not completely overlapping with those where HMGA1 and HEYL anti-correlate (Supplementary Fig. 10a).For example, they were not negatively correlated in lung SCC and the expression of these two genes was not prognostic of patient survival (Supplementary Fig. 10b,c).However, kidney renal clear cell carcinoma showed the strongest negative correlation between HMGA1 and HEY1 and their expression patterns were indicative of prognosis (Supplementary Fig. 10a,d,e).Together, these data demonstrate that an anti-correlation between HMGA1 expression and NOTCH1 activity is evident in cancer and that this correlation can be prognostic of patient outcome. ", "section_name": "NOTCH signalling represses the expression of HMGA genes.", "section_num": null }, { "section_content": "In the current study, we provide evidence for NOTCH-mediated 'lateral modulation' of chromatin structure at the microscopic and nucleosome scales.While RIS cells form prominent SAHFs at the microscopic scale 13,42 , at the nucleosome scale we observed a robust increase in chromatin accessibility.Both SAHFs and RIS- ", "section_name": "Discussion", "section_num": null }, { "section_content": "Fig. 8 NOTCH1 signalling mediates non-cell autonomous regulation of chromatin structure at the microscopic and nucleosome scale.Lateral induction of NOTCH1 activity in a signal-receiving cell by JAG1 on the surface of an adjacent cell (including cancer cells) can drive NIS.NIS cells form unique chromatin-accessible regions and microscopically 'smoothened' chromatin.In the context of RIS, non-cell autonomous activation of NOTCH1 signalling can repress the formation of AT-rich RASdriven accessible regions at the nucleosome level and SAHF formation at the microscopic level.Mechanistically, N1ICD represses HMGA1, which is responsible for SAHF formation and at least partially for the formation of ectopic-accessible chromatin in RIS cells driven accessibility can be inhibited by N1ICD-mediated repression of HMGA1 (Fig. 8).While the essential and structural role for HMGA1 in SAHF formation is well established 16 , its role in chromatin accessibility is unclear.HMGA proteins compete with Histone-H1 for linker DNA and thus affect chromatin compaction, as demonstrated by techniques such as fluorescence recovery after photo bleaching and MNase digestion assays 43,44 .Our data, using sequencing technology, demonstrate that HMGA1 effects the formation of ectopic accessible regions, potentially by facilitating the binding of other transcription factors, such as C/EBPβ, identified here through motif analysis of RIS-driven accessible regions.It is known that chromatin accessibility is an indicator of developmental maturity 45 and that cancer cells acquire ectopic accessible regions 45,46 .For example, during the metastasis of small cell lung cancer, a dramatic increase in chromatin accessibility at distal regulatory elements allows tumour cells to co-opt pre-programmed gene expression programmes, providing a growth advantage 32 .Thus our data raise a possibility that HMGA1 can drive pluripotency and cancer in part by modulating chromatin accessibility.It will be important to understand how HMGA1 facilitates both chromatin 'opening' at the nucleosome scale and the formation of SAHFs and to determine whether the two are related.We wonder whether the subset of HMGA1-dependent regions that are gene distal could have structural rather than regulatory functionality. Chromatin accessibility was also increased in NIS cells although these were often at distinct loci.Unlike RIS cells, NIS cells do not form SAHFs and are instead characterised by chromatin 'smoothening'.The mechanisms of chromatin smoothening and formation of NIS-driven accessible regions, and whether these events are related, remains unclear.Note, although knockdown of HMGA1 blocked formation of SAHFs and many RISdriven accessible regions, it was not sufficient to induce NIS-like chromatin smoothening or NIS-like chromatin accessibility, thus NOTCH signalling modulates chromatin by both HMGA1dependent and -independent mechanisms (Fig. 5b,c).One possible mechanism through which NOTCH modulates chromatin is through directed histone acetylation [47][48][49][50] .N1ICD activates gene transcription by recruiting histone acetyl-transferases 11 and was more recently shown to drive rapid and widespread deposition of H3K56ac 51 , which is known to be associated with nucleosome assembly, particularly in DNA replication and repair 52 . NOTCH signalling can be transiently activated during stressinduced senescence (e.g.oncogene-and DNA damage-induced senescence) 9 but also plays important roles during development and in cancer, thus 'lateral induction' of NOTCH activity through JAG1 could affect chromatin structure in various biologically relevant scenarios involving epithelial and/or fibroblast cells.Here we extend our analysis to the more specific 'epithelial-fibroblast' scenario that might mirror the cancer microenvironment where epithelial tumour cells are in active communication with stromal cells through the NOTCH1-JAG1-HMGA1 signalling axis.Consistently, using the Pten-null mouse model of prostate cancer, Su and colleagues 53 demonstrated that JAG1 expression in tumour cells facilitates the formation of a 'reactive stroma', which plays an important role in tumour development.It will be important to test whether chromatin structure is altered in the stroma of such tumours and whether this is dependent on HMGA1 repression.In NOTCH-ligand-expressing tumours, targeting chromatin-modifying enzymes in the stromal compartment may present a unique therapeutic opportunity to alter the tumour niche. ", "section_name": "SAHF positive", "section_num": null }, { "section_content": "Cell culture.IMR90 HDFs (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM)/10% foetal calf serum (FCS) in a 5% O 2 /5% CO 2 atmosphere. hTERT-RPE1 cells (ATCC) were grown in DMEM-F12/10% FCS in a 5% O 2 /5% CO 2 atmosphere.MCF7, H1299, A549 and Hep3B cells (ATCC) were grown in DMEM/10% FCS in a 5% CO 2 atmosphere.Cell identity was confirmed by STR (short tandem repeats) genotyping.Cells were regularly tested for mycoplasma contamination and always found to be negative. Co-cultures were set-up at a cell number ratio of 1:1 and performed in DMEM/ 10% FCS in a 5% O 2 /5% CO 2 atmosphere.For transwell experiments, IMR90 cells were plated in the bottom chamber and hTERT-RPE1 or tumour cells were plated into the top chamber of a Corning 12-well Transwell plate (CLS3460 Sigma). The following compounds were used in cultures: 100 nM 4-hydroxytamoxifen (4OHT) (Sigma), 10 µM DAPT (Sigma), 100 µM etoposide (Sigma), between 10 and 1000 ng/mL doxycycline (DOX) (Sigma) as indicated in individual figures. Vectors.The following retroviral vectors were used: pLNCX (clontech) ER: HRAS G12V30 ; pWZL-hygro for N1ICD-FLAG (residues 1758-2556 of human NOTCH1 9 ) and mRFP1; pLPC-puro for dnMAML1-mVenus (residues 12-74 of human MAML1) 9 , mRFP1, rThy1-mRFP1, JAGGED1-mVenus and mVenus; pQCXIH-i for DOX-inducible N1ICD-FLAG 9 ; MSCV-puro for miR30 shHMGA1 (shHMGA1 target sequence 5′-ATGAGACGAAATGCTGATGTAT-3' 16 ); and pCLIIPi 54 for pCLIIPi JAGGED1-mVenus. To generate pLPC-puro rThy1-mRFP1, we first PCR cloned mRFP into pLPCpuro (pLPC-puro-x-mRFP, where x denotes cloning sites to express mRFP-fusion proteins).The CDS of rat-Thy1 was PCR amplified from cDNA (a gift from M. de la Roche, CRUK CI, UK), removing the stop codon, before cloning into pLPCpuro-x-mRFP.To generate pLPC-JAGGED1-mVenus, the CDS of human JAGGED1 was amplified using cDNA derived from N1ICD-expressing IMR90 cells, removing the stop codon, before cloning into pLPC-puro-x-mVenus.To generate pCLIIPi (DOX-inducible) JAG1-mVenus, JAGGED1-mVenus was subcloned using PCR into pCLIIPi. Flow cytometry.Analysis of ectopic JAG1-mVenus expression was conducted by flow cytometry 9 .Cells were fixed using 4% paraformaldehyde (PFA) in phosphatebuffered saline (PBS) and stained with anti-JAG1-APC (FAB1726A, R&D Systems, 1:10) or isotype control antibody (IC0041A, R&D Systems, 1:10) before analysis on a FACSCalibur flow cytometer (Becton Dickenson).Flow data were further analysed using FlowJo v10. MACS and FACS.MACS of rThy1-expressing cells was performed using CD90.1 microbeads (130-094-523, Miltenyl Biotec) according to the manufacturer's instructions.Fluorescence-activated cell sorting (FACS) was performed using an Influx (Becton Dickenson) flow cytometer. Fluorescence microscopy.Analysis was performed as previously described 16 .Briefly, cells were plated onto #1.5 glass coverslips the day before fixation to achieve approximately 60% confluence.Cells were fixed in 4% (v/v) PFA and permeabilised with 0.2% (v/v) Triton X-100 in PBS with DAPI.Coverslips were mounted onto Superfrost Plus slides (4951, Thermo Fisher) with Vectashield Antifade mounting medium (H-1000, Vector Laboratories Ltd.).Images were obtained using a Leica TCS SP8 microscope with a HC PL APO CS2 1.4NA 100× oil objective (Leica Microsystems).At least 30 nuclei were captured per biological replicate and condition before Fiji 55 was used to calculate nuclear area, standard deviation and maximum intensity of DAPI signal per nucleus.Specifically, the DAPI channel was duplicated, desaturated and a threshold applied using the Otsu method before holes were filled and the 'analyse particles' function was used to create a region of interest per nucleus for measurement in the original DAPI-stained image.SADS were visualised by DNA-fluorescence in situ hybridisation as previously described 15 using fluorescent probes that target the α-satellite repeat sequence (5′-CTTTTGATAGAGCAGTTTTGAAACACTCTTTTTGTA-GAATCTGCAAGTGGATATTTGG-3′).The percentage of SAHF-and SADSpositive cells was counted by scoring at least 200 cells per replicate and condition. Quantitative reverse transcription-PCR.RNA was prepared using the Qiagen RNeasy Plus Kit (74136, Qiagen) according to the manufacturer's instructions and reverse-transcribed to cDNA using the Applied Biosystems High-Capacity Reverse Transcription Kit (43-688-13, Thermo Fisher).Relative expression was calculated as previously described 16 on an Applied Biosystems Quantstudio 6 by the 2 -ΔΔCt method 56 using β-actin (ACTB) as an internal control.The following primers were used: ACTB forward: 5′-GGACTTCGAGCAAGAGATGG-3′ ACTB reverse: 5′-AGGAAGGAAGGCTGGAAGAG-3′ HEYL forward: 5′-CTCCAAAGAATCTGTGATGCCAC-3′ HEYL reverse: 5′-CCAGGGACAATGAAAGCAAGTTC-3′ HEY1 forward: 5′-CCGCTGATAGGTTAGGTCTCATTTG-3′ HEY1 reverse: 5′-TCTTTGTGTTGCTGGGGCTG-3′ HES1 forward: 5′-ACGTGCGAGGGCGTTAATAC-3′ HES1 reverse: 5′-ATTGATCTGGGTCATGCAGTTG-3′ HMGA1 forward: 5′-GAAAAGGACGGCACTGAGAA-3′ HMGA1 reverse: 5′-TGGTTTCCTTCCTGGAGTTG-3′ HMGA2 forward: 5′-AGCGCCTCAGAAGAGAGGA-3′ HMGA2 reverse: 5′-AACTTGTTGTGGCCATTTCC-3′ Protein quantification by immunoblotting.Immunoblotting was performed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels using the following antibodies: anti-β-actin (Sigma, A5441, 1:10,000); anti-HRAS (Calbiochem, OP-23, 1:500); anti-NOTCH1 (Cell Signaling, 4380, 1:500); anti-HES1 (Cell Signalling, 11988, 1:1000); anti-FLAG (Cell Signaling, 2368, 1:1000), anti-HMGA1 (Cold Spring Harbor Labs, #37, 1:1000); anti-HMGA1 (Abcam, Ab4078, 1:1000); anti-HMGA2 (Cold Spring Harbor Labs, #24, 1:1000); anti-GFP (Clontech 632377, 1:1000); and anti-JAG1 (Cell Signaling, 2155, 1:1000).Images of uncropped immunoblots are included in Supplementary Fig. 11. ATAC-seq.ATAC-seq samples were generated as previously 31 using 100,000 IMR90 cells and 13 cycles of PCR amplification.Samples were size selected between 170 and 400 bp (in order to isolate 'nucleosome free' and 'mono-nucleosome' fragments) using SPRIselect beads (B23319, Beckman Coulter) before single-end sequencing to generate 75 bp reads on the NextSeq-500 platform (Illumina). ChIP-seq.Chromatin immunoprecipitation (ChIP) was performed as previously described using 20 µg of sonicated chromatin 57 from growing and RIS IMR90 ER: HRAS G12V cells and 5 µg of anti-H3K27ac antibody (Clone CMA309 58 ) or 5 µg of H3K4me1 antibody (Clone CMA302 58 ).Libraries were prepared using the NEB-Next Ultra II DNA Library Prep Kit for Illumina (37645, New England Biolabs) according to the manufacturer's instructions except that size selection was performed after PCR amplification using SPRIselect beads (B23319, Beckman Coulter).Samples were sequenced single-end using 50 bp reads on the HiSeq-2500 platform (Illumina). RNA-seq.RNA-seq data was generated from IMR90 ER:HRAS G12V cells expressing a short-hairpin targeting the 3′ UTR of human HMGA1 (RIS+shHMGA1).RNA was purified as above and quality checked using the Bioanalyser eukaryotic total RNA nano series II chip (Agilent).mRNA-seq libraries were prepared from six biological replicates of each condition using the TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer's instructions and sequenced using the HiSeq-2500 platform (Illumina). Generation of genome-edited JAG1 knockout clones.The following CRISPR guides were designed against Exon 2 of JAG1 (NM_000214.2) (Supplementary Fig. 7d): sgJAG1_2.1:5′-AGTCCCGCGTCACGGCCGGG-3′ (PAM:GGG) and sgJAG1_2.2:5′-CGCGGGACTGATACTCCTTG-3′ (PAM:AGG).Oligonucleotides (Sigma Aldrich) were cloned into pSpCas9(BB)-2A-GFP 59 .pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138).Guide cutting efficiency was determined in A549 cells using the T7 assay (New England Biolabs, following manufacturer's instructions).To generate independent, non-sister clonal cell lines, A549 cells were transiently transfected (Lipofectamine 3000, Thermo Fisher Scientific) with PX458-empty (control), PX458-sgJAG1_2.1 and PX458-sgJAG2.2,and single cell was cloned 96 h posttransfection by FACS (BD FACSAria II).gDNA was extracted from each clone (Extracta DNA Prep, VWR, 95091-025) and Exon 2 of JAG1 was amplified by PCR (FastStart HF System (Sigma Aldrich, 3553361001)) using the following primers (universal Fluidigm tag in lower case, JAG1-specific sequence in upper case)): Forward: 5′-acactgacgacatggttctaca-GAGCTGCAGAACGGGAACT-3′; Reverse: 5′-tacggtagcagagacttggtct-CTTGAGGTTGAAGGTGTTGC-3′.Amplicons were diluted 1:150 and re-amplified with Fluidigm barcoding primers (incorporating a unique sample barcode and Illumina P5 and P7 adapter sequences), pooled and subjected to sequencing (Illumina MiSeq platform).The AmpliconSeq analysis pipeline was used for data processing and variant calling.Briefly, reads were aligned against the reference genome (GRCh38) using BWA-MEM 60 and variants were called using two methods (VarDict 61 and GATK HaplotypeCaller 62 ).Consensus variants and their effects on CRISPR clones were then calculated.All clones used in this paper were STR genotyped and confirmed as free from mycoplasma. RNA-seq analysis.Reads were mapped to the human reference genome hg19 with the STAR (version 2.5.0b)aligner 63 .Low-quality reads (mapping quality <20) as well as known adapter contaminations were filtered out using Cutadapt (version 1.10.0) 64.Read counting was performed using Bioconductor packages Rsubread 34 and differential expression analysis with edgeR 33,34 .The conditions were contrasted against the growing samples.Genes were identified as differentially expressed with a FDR (false discovery rate) cut-off of 0.01 and an absolute value of logFC (log 2 of the fold change) >0.58. ChIP-seq and ATAC-seq analysis.ChIP-seq and ATAC-seq reads were mapped to the human reference genome (hg19) with BWA (version 0.7.12) 60 .Low-quality reads (mapping quality <20) as well as known adapter contaminations were filtered using Cutadapt (version 1.10.0) 64, and reads mapping to the 'blacklisted' regions identified by ENCODE 65 were further removed.Average fragment size was determined using the ChIPQC Bioconductor package 66 , and peak calling was performed with MACS2 (version 2.1.0) 67, using fragment size as an extension size (--extsize) parameter.High-confidence peak sets for each condition were identified separately using only those peak regions that were present in at least two replicates.Differential accessibility analysis.THOR 35 and edgeR 33,34 were used to identify differentially accessible regions between conditions.For the comparisons 'NIS vs. growing' and 'RIS vs. growing', the intersect of regions detected by THOR and edgeR was taken.This approach gave us a robust set of regions that are altered in RIS and NIS conditions.For other comparisons where volcano plots were generated, edgeR was used to interrogate how different genetic and cell-culture manipulations effect the alterations detected in RIS. edgeR 33,34 was used on a merged set of growing, RIS, NIS, N+RIS, shHMGA1 and RIS/shHMGA1 high-confidence ATAC-seq peak sets (present in at least two replicates of a single condition) to identify regions of differential accessibility between conditions.We utilised the TMM method implemented in edgeR for normalisation and dispersion calculation of the replicated samples.The results were further filtered based on FDR < 0.05 and logFC ≥ 0.58 or ≤-0.58. THOR is a Hidden Markov Model based approach that utilises all mapped reads and identifies the differentially accessible regions between two conditions.THOR was used in parallel with edgeR to identify the differences between the conditions using our pre-computed normalisation factors (see section 'Generation of normalised coverage files') to normalise between samples.Regions were further filtered using a -log(p-value) cut-off of 10. Annotating differentially accessible regions.Bedtools intersect (v2.26.0) 68 was used to identify regions annotated as 'unchanged' by extracting high-confidence peaks in the 'growing' condition that did not intersect with regions that become more or less accessible in the NIS and/or RIS conditions (relative to the growing condition).The rest of the categories (open or closed in RIS or NIS) were identified based on the differential accessibility analysis compared to growing using the intersect of THOR and edgeR as described above.Regions displaying altered chromatin accessibility were mapped to genomic annotations or repeat regions using bedtools v2.26.0 68 .For the genomic annotations, we used TSSs from the FANTOM database 69 , repeats from repeatMasker (UCSC genome browser) and other genomic features (exons, introns, UTRs, etc.) were extracted from the UCSC Table Browser.The enhancers were identified based on our own H3K4me1 and H3K27ac histone mark ChIP-seq data sets; all regions that had peaks in both of these marks in either growing or RIS cells were considered as enhancers. Intersecting consensus peaks and generating Venn diagrams.The Homer (v3.12) 70 command 'mergePeaks' with default settings and the output options '-venn' and '-prefix' were used to generate values for plotting Venn diagrams and associated bed files for further analysis.Only literal overlaps (overlapping by 1 bp) were considered.Venn diagrams were plotted using the R package 'Venneuler' (https://cran.rproject.org/web/packages/venneuler/index.html). Calculating proximity to genes and GC percentage.To calculate the distance of consensus peaks from TSSs and GC percentage of accessible regions, the Homer (v3.12) 70 command 'annotatePeaks.pl'was used with default settings and the output option '-CpG'. Gene enrichment analysis.Altered accessible regions within 500 bp of a gene TSS were identified using Homer as described above.Gene enrichment analysis was performed using the GO Biological Process 2015 annotation provided on the webtool 'Enrichr' 71 (http://amp.pharm.mssm.edu/Enrichr/).Generation of normalised coverage files.A previously described approach was used to generate scaling factors for each ATAC-seq condition relative to others 32 .Briefly, we reasoned that the enrichment of reads within ATAC-seq peaks containing TSSs of genes that are both expressed (logCPM > mean logCPM) and have low variance between conditions (-0.14 < logFC < 0.14) by RNA-seq should not vary, unless there are differences in ATAC-seq sample quality, preparation or sequencing.By reanalysing our previously published IMR90 RNA-seq data 9 together with newly generated RNA-seq samples for RIS+shHMGA1 cells, we identified 589 genes that fit these criteria.We counted the reads from the ATACseq samples that map to these specific genes using Rsubread 34 and computed scaling factors based on the mean counts for each condition separately.Normalised coverage files (bigWig) were generated by pooling reads from all of the replicates and applying the calculated scaling factors using the 'genomecov' function in bedtools, sorting the resulting normalised bedGraph files and then converting them to bigWigs using the 'bedGraphToBigWig' function from UCSC. Generation of clustered heatmaps.Heatmaps were generated using normalised coverage of peaks (+/-2.5 kb) representing novel accessible regions (regions with significantly altered chromatin accessibility in RIS or NIS relative to growing cells) with k-means clustering using the deepTools package 72 . PCA analysis and correlation heatmaps.Samples were normalised with the precalculated normalisation factors (as described above in 'Generation of normalised coverage files'), and reads from all growing, RIS, NIS, N+RIS, shHMGA1 and RIS/ shHMGA1 consensus peak sets (present in at least two replicates across all of the samples) were extracted and used in the PCA analysis and the correlation analysis of data sets.Pearson correlation was calculated between samples based on these normalised read counts and correlation heatmaps were generated with pheatmap (http://CRAN.R-project.org/package=pheatmap) and WPGMA clustering.PCA plots were generated using ggplot2 73 . Reads from publicly available ATAC-seq and DNase-seq data sets [74][75][76][77] (references and NIH Epigenomics Roadmap Initiative) were extracted from the same regions; however, since these were not included in normalisation factor calculation, standard CPM normalisation was used for Supplementary Fig. 4c. Volcano plots.edgeR calculated statistical parameters (logFC and logFDR) were used to visualise differentially accessible regions in RIS (red) and NIS (purple) compared to growing cells in the comparisons indicated.Plots were generated using ggplot2 73 . Motif enrichment analysis.Meme-ChIP suite (version 4.12.0),together with Hocomoco (version 11) human and mouse PWMs, was used to detect motif enrichment in a 600 bp region centred at the peak summit. TCGA analysis.We analysed the expression levels of NOTCH-associated genes in the publicly available RNA sequencing data generated by the TCGA Research Network: http://cancergenome.nih.gov/ 41. Computational analysis and statistical testing of the Next-Generation Sequencing data was conducted using the R statistical programming language 78 .Filtered and log 2 -normalised RNA expression data along with all available clinical data were downloaded from the GDAC firehose database (run: stddata_2015_06_01) for each gene of interest from the relevant cancer-specific collections. Correlation testing for associations between expressed genes was performed using the cor.test function in R to calculate the Pearson's product moment correlation coefficient and test for significant deviation from no correlation.Plotting of TCGA data was performed using the ggplot2 R package 73 .Survival analysis was performed using the survminer and survival 79 R packages.Kaplan-Meier estimated survival curves were constructed using the TCGA clinical data.Statistical testing of differences between survival curves used the G-rho family of tests, as implemented in the survdiff function of the survival package. ", "section_name": "Methods", "section_num": null }, { "section_content": "No statistical method was used to predetermine sample size and experiments were not randomised.Statistical analyses were conducted using the Graphpad Prism 6 and R statistical software, except for TCGA data analysis (which was as described in the methods above).One-way analysis of variance with Tukey's correction for multiple comparisons was used for data sets with >2 conditions.Two-sample t-tests were used for two-condition comparisons.The statistical tests were justified as appropriate based on the number of samples compared and the assumed variance within populations.A p-value of <0.05 was used to indicate statistical significance. ", "section_name": "Statistics and reproducibility.", "section_num": null } ]	[ { "section_content": "We thank all members of the Narita laboratory for helpful discussions, M. de la Roche for reagents and staff of the Cancer Research UK Cambridge Institute core facilities for technical support.The University of Cambridge, Cancer Research UK and Hutchison Whampoa supported this work.M.N., S.B. and I.A.R laboratories are funded by a Cancer Research UK Cambridge Institute Core Grant (C14303/A17197).M.N. is also supported by a Cancer Research UK Early Detection Pump Priming award (C20/A20976), Medical Research Council (MR/M013049/1) and Tokyo Tech World Research Hub Initiative (WRHI).M.H. is supported by a CRUK Clinician Scientist Fellowship (C52489/A19924).R.H.-H. is funded by an EMBO Long-Term fellowship.S.A.S. and D.B. were supported by Medical Research Council core funding.H.K. was supported by JSPS KAKENHI JP25116005, JP26291071, 15K21730 and 17H01417. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "Data availability.The RNA-seq, ChIP-seq and ATAC-seq data generated for this study have been deposited at the Gene Expression Omnibus (GEO) with the accession number GSE103590.Gene expression data from RIS, NIS and N+RIS is previously published 9 and available under GEO accession number GSE72404. ", "section_name": "", "section_num": "" }, { "section_content": "Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-018-04283-9. Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/ Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Author contributions", "section_num": null }, { "section_content": "", "section_name": "Author contributions", "section_num": null }, { "section_content": "Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-018-04283-9. Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/ Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Additional information", "section_num": null } ]
10.18632/oncotarget.7879	Metformin combined with sodium dichloroacetate promotes B leukemic cell death by suppressing anti-apoptotic protein Mcl-1	Metformin and the mitochondrial targeting dichloroacetate (DCA) have recently received attention due to their ability to inhibit anaerobic glycolysis, which renders most cancer cells resistant to apoptosis induction. We observed that Metformin alone exhibited a dose-dependent anti-leukemic activity in both B leukemic cell lines and primary B-chronic lymphocytic leukemia (B-CLL) patients' cells and its anti-leukemic activity was enhanced when used in combination with DCA. In order to overcome the problems of poor bioavailability and cellular uptake, which limit DCA efficacy, we have designed and synthetized cocrystals consisting of Metformin and DCA (Met-DCA) at different stoichiometric ratios. Of note, the MetH(2)(++)•2DCA(-) cocrystal exhibited enhanced in vitro anti-leukemic activity, with respect to the treatment with the mix consisting of Metformin plus DCA. In particular, the treatment with the cocrystal MetH(2)(++)•2DCA(-) induced a synergistic apoptotic cell death coupled to a marked down-modulation of the anti-apoptotic Mcl-1 protein. Taken together, our data emphasize that innovative compounds based on Metformin-DCA combination merit to be further evaluated as chemotherapeutic agents for the treatment of B-CLL.	[ { "section_content": "Most cancers are characterized by enhanced glycolytic flux for ATP production, enhanced glucose to lactate conversion and reduced mitochondrial oxidative phosphorylation, even under aerobic conditions [1][2][3].Interestingly, this cancer-specific metabolic remodeling can be reversed by dichloroacetate (DCA), a mitochondriatargeting small molecule able to penetrate most tissues after oral administration [4].DCA is a generic drug with low price, which has been used for human treatments for more than 30 years and has received renovated attention because of interesting preclinical antitumoral characteristics, assessed in solid tumor cell lines [4][5][6][7], and relatively low toxicity on normal cells.Some recent studies of our and other groups have demonstrated that DCA is also effective against hematological malignancies, such as multiple myeloma [8] and in particular, for the purpose of this study, against B-chronic lymphocytic leukemia (B-CLL) cells [9,10]. Among the great variety of anti-cancer drugs, another inexpensive and safe drug, which has recently revealed a potential anti-leukemic activity, is Metformin [11][12][13][14].This molecule represents the most commonly prescribed drug for type 2 diabetes mellitus [11].Anyhow, in recent years, multiple lines of evidence have provided support for the hypothesis that treatment with Metformin results in decreased incidence, progression, and mortality of different human cancers.Moreover, a number of in vitro studies have documented the antiproliferative, anti-invasive, and antimetastatic effects of Metformin in multiple cancer cell types [15][16][17][18].Interestingly, DCA and Metformin share several mechanisms, potentially involved in their anticancer activity, by disrupting mitochondrial respiratory chain complex and decreasing the ATP synthesis [19]. On these bases, the aim of the present study was to evaluate the potential therapeutic perspectives of Metformin plus DCA as innovative anti-leukemic drug combination.Herein, we have evaluated the in vitro effects of Metformin used alone and in combination with DCA on B-leukemic cells, including primary B-CLL patient cells, by assessing cell viability, cell cycle progression, apoptosis, as well as the expression of apoptotic signaling modulators.Of note, to improve the efficacy of the drug combination, we have designed, synthetized and functionally validated Metformin-DCA cocrystals. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "In the first set of experiments, we have evaluated the in vitro effect of Metformin on B leukemic cell lines (EHEB and JVM-2), as well as on primary B-CLL patient cell samples.All leukemic cells were characterized by having a p53 wild-type status, a feature typical of the majority of the B-CLL at diagnosis [20][21][22][23][24][25].Treatment with Metformin exhibited a dose-and time-dependent cytotoxicity on both B-leukemic cell lines (Figure 1A) as well as on B-CLL patient cell cultures (Figure 1B).Of note, the IC 50 mean values (±SD) calculated after 48 hours of treatment in B leukemic cell lines (11.58±0.77mM) and B-CLL patient derived cell cultures (10.17±1.04mM) were comparable. ", "section_name": "Metformin promotes cytotoxicity in B leukemic cell lines and in primary B-CLL cells", "section_num": null }, { "section_content": "Starting from our recent studies documenting anti leukemic activity of DCA towards B-CLL [9,10], in the next group of experiments we have explored the potentiality of using Metformin in combination with DCA.B-CLL cells were treated with Metformin and DCA (used in the range of 0.1-20 mM) as single agents and in combination.In particular, leukemic cells were treated with serial concentrations of Metformin and DCA at a constant Metformin:DCA ratio (either 1:1 or 1:2) for data analysis by the method of Chou and Talalay [26].Combined treatment with Metformin plus DCA, at 1:2 ratio, resulted in significantly (p<0.05)enhanced cytotoxicity with respect to the single agents in both B leukemic cell lines as well as in primary B-CLL patient samples (Figure 2A), with a synergistic effect (Figure 2B) documented by an average Combination Index (CI) value <1.On the other hand, no significant cytotoxicity was observed in normal peripheral blood cells exposed to the single drugs, confirming literature data [9,27], as well as to the Metformin plus DCA combination (Figure 2A).Starting from these results and considering that DCA molecule exhibits poor bioavailability and cellular uptake, we have synthetized new molecules consisting of cocrystals of Metformin and DCA in different stoichiometric ratios: MetH + •DCA -(1:1; Figure 3A) and MetH 2 ++ •2DCA -(1:2; Figure 3B).When tested on leukemic cell lines and B-CLL patient derived primary cells, these compounds exhibited in vitro anti-leukemic activity.In particular, maximal cytotoxic effects were observed when cell cultures were treated with the cocrystal MetH 2 ++ •2DCA - (from now on, named Met-DCA cocrystal), which exhibited an enhanced (p<0.05)activity with respect to the treatment with a mix of the two reference drugs, used at the appropriate concentrations (Figure 4).Analysis of the cell cycle profile (Figure 5A) revealed that the cytotoxicity induced by the treatment with the Met-DCA cocrystal was effective in increasing the cytostatic effect induced by DCA (id, accumulation in G1 phase and reduction in S phase of the cell cycle).Moreover, the Met-DCA cocrystal was particularly effective as pro-apoptotic molecule resulting in a significant (p<0.05)increase of the degree of apoptosis with respect to the treatment with Metformin and DCA used as single agents (Figure 5B-5C).Of note, the pro-apoptotic effects of the Met-DCA cocrystal were significantly (p<0.05)higher also with respect to the treatment with the mix of Metformin plus DCA (Figure 5B-5C). ", "section_name": "Anti-leukemic activity of Metformin plus DCA and of Met-DCA cocrystals", "section_num": null }, { "section_content": "In order to evaluate the molecular basis explaining the antitumoral effects of the Met-DCA cocrystal, after in vitro treatments, we have analyzed the expression profile of intracellular effectors known to stimulate/suppress leukemic cell survival.In particular, considering previous studies on the molecular effects of Metformin, alone or in combination with other therapeutic compounds, in solid tumor cell models and in multiple myeloma cells [28][29][30], we have first investigated the expression levels of Mcl-1.Of note, Mcl-1 is one of the most important anti-apoptotic Bcl-2 family member, which induces CLL cell survival and therefore is associated with CLL response to the treatments and disease progression [31,32].As shown in Figure 6, in our experimental models, Mcl-1 protein levels were significantly decreased upon 24 hours of treatment with the combination of Metformin plus DCA.Of interest, maximal down-regulation was observed by treatment with the Met-DCA cocrystal (Figure 6).It has to be underlined that the down-modulation of Mcl-1, coupled to the induction of apoptosis, in response to Met-DCA cocrystal was documented not only in B leukemic cells with a p53 wild-type status, but also in the B lymphoblastoid cell line MEC-2 harboring mutated p53 (Figure 7).Conversely, the levels of the pro-apoptotic Bcl-2 protein did not change upon drug treatments neither in p53 wild-type (Figure 6) nor in p53 mutated cells (Figure 7). In the next experiments, we have analyzed Mcl-1 protein levels at early time points upon drug exposure.As shown in Figure 8A, Mcl-1 down-modulation was evident already after 3-5 hours of treatment with the Met-DCA cocrystal, well before the onset of apoptosis.In parallel, since in different cancer cells Mcl-1 is induced by Akt and STAT-3 intracellular pathways [33][34][35], which are implicated in B-CLL pathogenesis [12,[36][37][38][39], we have investigated the potential involvement of Akt and STAT-3 in the anti-leukemic activity of Met-DCA cocrystal.As shown in Figure 8B, down-modulation of Mcl-1 in response to the Met-DCA cocrystal was coupled to a significant down-regulation of phospho-Akt, total Akt, and phospho-STAT3 protein levels.On the other hand, in line with previous studies on Metformin [13,14,18], our present study documented the ability of Met-DCA cocrystal of stimulating the AMP-activated protein kinase (AMPK) pathway (id, AMPK phosphorylation; Figure 8C), an energy-sensing system associated with Mcl-1 down-modulation in different cell models [40][41][42]. ", "section_name": "The Met-DCA cocrystal-induced cell death is accompanied by Mcl-1 down-regulation", "section_num": null }, { "section_content": "Based on FDA definition, cocrystals are solids composed of two or more molecules in the same crystal lattice held together in stoichiometric amounts by freely reversible, noncovalent forces of interaction [43].When one of the molecules is an Active Pharmaceutical Ingredient (API) they are called pharmaceutical cocrystals and have received recent attention because of better pharmaceutical profiles over the native drugs [44].Moreover, the concept of modifying the properties of a single API by cocrystallization with a coformer, that may be another API that improves the delivery and complements the efficacy of the reference drug, has receiving increasing interest for drug development [45]. In virtue of our previous observations documenting the cytotoxic properties of DCA towards B-CLL [9,10], in the present in vitro study, we have analyzed the potential antileukemic activity of Metformin used in combination with DCA, either as a mix of the two compounds or as a cocrystal composed of Metformin and DCA (1:2).We have documented the ability of the Metformin and DCA combination and, even more, of the Met-DCA cocrystal, in promoting cytotoxicity in both B leukemic cell lines and B-CLL patient derived cell cultures as result of both a cytostatic and pro-apoptotic effect. Beyond the plausible effects on several physicochemical properties (such as solubility), we observed that the use of the Met-DCA cocrystal enhanced the biological effect of the mix composed by the two drugs, probably as a result of an improved cellular uptake of the cocrystal compared to the reference drugs.We could argue that the molecule Metformin may act as a carrier for DCA, which indeed requires high dosage in therapies because of the low bioavailability [46].The concept of an enhanced bioavailability of the cocrystal, associated to the advantage of administration of a single pharmaceutic compound instead of the separated reference drugs, certainly offers better opportunities for future in vivo preclinical assessments (i.e., investigations in animal models) and for the translation of the Met-DCA, compound based on two inexpensive and safe drugs, to clinical applications. In the effort to elucidate the molecular mechanism of action of Met-DCA cocrystal, we investigated whether the anti-apoptotic Bcl-2 family members might represent targets for Met-DCA cocrystal.Our data documented a correlation between Mcl-1 down-regulation and cytotoxicity induced by the Metformin plus DCA combination and Met-DCA cocrystal in B leukemic cells, with an early downregulation of Mcl-1 (but not of Bcl-2), associated to a concomitant down-regulation of phospho-Akt, total Akt, and phospho-STAT3 protein levels, which anticipated the onset of apoptosis.These data are particularly interesting since alteration of the intrinsic pathway of apoptosis is a major target of cancer cells to evade apoptosis, and often this goal is achieved through the increase in the expression level of Bcl-2 and/or Mcl-1 [47].In this respect, several Bcl-2 inhibitors (such as navitoclax and venetoclax) have shown efficacy as chemotherapy agents in clinical trials, by binding with high affinity Bcl-2 and Bcl-Xl [48,49].However, none of these compounds can bind and antagonize the prosurvival activity of the Mcl-1 Bcl-2 family member, which promote the development of many cancers and/or in their resistance to chemotherapy [50].It is also a key factor in the resistance of malignant cells to navitoclax and venetoclax.Interestingly, it has been recently shown that the Notch-dependent upregulation of Mcl-1 promotes survival of B-CLL [51] and thus it represents an axis for therapeutic interventions in the perspective to eradicate the leukemic clone. Since Mcl-1 silencing has been found to elicit tumor regression and cell death in various cancer models [52], including B-CLL [53], our data provide the rationale for further evaluating the use of Met-DCA cocrystal (also in combination with the Bcl-2 specific targeting drug venetoclax) for the treatment of B-CLL, also in virtue of the potential anti-leukemic activity of Met-DCA independently of the p53 status. ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "The B leukemic cell lines EHEB, JVM-2 and MEC-2 were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).EHEB and JVM-2 cell lines were routinely cultured in RPMI-1640, whereas MEC-2 cells were maintained in IMDM, all supplemented with 10% FBS, L-glutamine and Penicillin/streptomycin (all from Gibco, Grand Island, NY).For experiments with primary cells, peripheral blood samples were collected in heparin-coated tubes from either normal blood donors or from B-CLL patients following informed consent, in accordance with the Declaration of Helsinki and in agreement with institutional guidelines (University-Hospital of Ferrara).The diagnosis of B-CLL was made by peripheral blood morphology and immunophenotyping and all patients had been without prior therapy at least for three months before blood collection.Peripheral blood mononuclear cells (PBMC) were isolated by gradient centrifugation with lymphocyte cell separation medium (Cedarlane Laboratories, Hornby, ON).T lymphocytes, NK lymphocytes, granulocytes and monocytes were negatively depleted from peripheral blood leucocytes (PBL) with immunomagnetic microbeads (MACS microbeads, Miltenyi Biotech, Auburn, CA), with a purity >95% of resulting CD19 + B-CLL population, assessed by flow cytometry, as previously described [54,55].For in vitro assays, both freshly isolated or thawed B-CLL cells, previously resuspended in freezing solution (10% DMSO and 90% FBS) and cryopreserved in liquid nitrogen, were seeded in RPMI containing 10% FBS, L-glutamine and Penicillin/Streptomicin (Gibco).Freezing/ thawing of B-CLL primary cells did not influence sensitivity to the treatments, since we assessed in parallel fresh and frozen/thawed cells of three patients without observing any significant cytotoxic differences. ", "section_name": "Cell cultures and treatments", "section_num": null }, { "section_content": "Leukemic cells were treated with serial doses of Metformin hydrochloride (1,1-Dimethylbiguanide hydrochloride, Sigma-Aldrich, St Louis, MO; range 0.1-10 mM), DCA (Sigma-Aldrich; range 0.2-20 mM), used alone or in combination, or with a Met-DCA cocrystal that was synthesized to combine Metformin plus DCA. At different time points after treatment, cell viability was examined by Trypan blue dye exclusion and MTT (3-(4,5-dimethilthiazol-2yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay (Roche Diagnostics Corporation, Indianapolis, IN) for data confirmation, as previously described [56,57].IC 50 values were calculated from dose-response curves constructed by plotting cell survival (%) versus drug concentration. Levels of apoptosis were quantified by Annexin V-FITC/propidium iodide (PI) staining (Immunotech, Marseille, France) followed by analysis using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA).To avoid non-specific fluorescence from dead cells, live cells were gated tightly using forward and side scatter, as described [58,59].The cell cycle profile was analyzed by flow cytometry after 5-bromodeoxyuridine (BrdU) incorporation, as described [60]. ", "section_name": "Culture treatments, assessment of cell viability, apoptosis and cell cycle profile", "section_num": null }, { "section_content": "All adducts of Metformin with DCA (Met-DCA 1:1, MetH + •DCA -; Met-DCA 1:2, MetH 2 ++ •2DCA -) were prepared using reagents and solvents purchased from a commercial source (Sigma Aldrich) and used without further purification.In particular, synthesis was carried out as follows: -Met-DCA 1:1 (MetH •2DCA -).Equimolar amounts of sodium dichloroacetate (512.8 mg, 3.4 mmol) and dichloroacetic acid (438,1 mg, 3.4 mmol) were added to a methanol solution (30 mL) of metformin hydrochloride (562.7 mg, 3.4 mmol) and stirred at room temperature.After 12 hours the solvent was evaporated under reduced pressure and 2-propanol (100 mL) was added to the solid sediment.Suitable crystals for X-ray diffraction were obtained by slow evaporation at room temperature of clear solutions of neutral metformin and dichloroacetic acid dissolved in 1:1 and 1:2 molecular ratio, respectively, in a 50:50 (v:v) methanol/n-pentanol mixture. The identity of chemical composition and crystal phase of the compounds obtained as powder in larger amount was assessed by comparing the experimental X-ray powder diffraction (XRPD) patterns with those calculated using the program Mercury [x2] from the structures determined by single-crystal X-ray diffraction.The crystal data of compounds Met-DCA 1:1 and Met-DCA 1:2 were collected at room temperature using a Nonius Kappa CCD diffractometer with graphite monochromated Mo-Kα radiation (Bruker-Nonius, Milan, Italy).[61,62]. ", "section_name": "Synthesis of the multicomponent Met-DCA cocrystals", "section_num": null }, { "section_content": "For Western blotting analysis, cells were lysed as previously described [63,64].Protein determination was performed by BCA Protein Assay (Thermo Scientific, Rockford, IL).Equal amounts of protein for each sample were migrated in SDS-polyacrylamide gels and blotted onto nitrocellulose filters.The following Abs were used: anti-Mcl-1 (S-19) and anti-Bcl-2 (100) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-AMPKα (Thr172, D79.5E), anti-AMPKα, anti-phospho-STAT3 (Ser727) and anti-STAT3 from Cell Signalling (Danvers, MA); anti-phospho-Akt1/PKBα (Ser473) from Merck Millipore (Darmstadt, Germany); anti AKT/PKBα from BD; anti-tubulin from Sigma-Aldrich.After incubation with antimouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary Abs (Sigma-Aldrich), specific reactions were revealed with the ECL Lightning detection kit (Perkin Elmer, Waltham, MA).The estimation of the densitometry values of bands was obtained by the ImageQuant TL software (GE Healthcare, Buckinghamshire, UK). ", "section_name": "Western blotting analyses", "section_num": null }, { "section_content": "The results were evaluated by using analysis of variance with subsequent comparisons by Student's t-test and with the Mann-Whitney rank-sum test.Statistical significance was defined as p<0.05.In order to investigate the effect of Metformin plus DCA combination, leukemic cells were treated with serial doses of Metformin or DCA, individually or in combination using a constant ratio (Metformin:DCA).Results were analyzed with the method of Chou and Talalay [26] to determine whether combined treatment yields greater effects than expected from summation alone: a combination index (CI) of 1 indicates an additive effect, while a CI below 1 indicates synergism.For this purpose cell viability data were analyzed with the CalcuSyn software (Biosoft, Cambridge, UK) and reported either as CI values or as doseeffect curves directly drawn by the CalcuSyn software. ", "section_name": "Statistical analysis and assessment of the effect of combination treatment", "section_num": null } ]	[ { "section_content": "All authors declare no conflict of interest. ", "section_name": "CONFLICTS OF INTEREST", "section_num": null } ]
10.3390/curroncol30050339	Impact of Fixed-Duration Oral Targeted Therapies on the Economic Burden of Chronic Lymphocytic Leukemia in Canada	<jats:p>Background: Continuous oral targeted therapies (OTT) represent a major economic burden on the Canadian healthcare system, due to their high cost and administration until disease progression/toxicity. The recent introduction of venetoclax-based fixed-duration combination therapies has the potential to reduce such costs. This study aims to estimate the prevalence and the cost of CLL in Canada with the introduction of fixed OTT. Methods: A state transition Markov model was developed and included five health states: watchful waiting, first-line treatment, relapsed/refractory treatment, and death. The number of CLL patients and total cost associated with CLL management in Canada for both continuous- and fixed-treatment-duration OTT were projected from 2020 to 2025. Costs included drug acquisition, follow-up/monitoring, adverse event, and palliative care. Results: The CLL prevalence in Canada is projected to increase from 15,512 to 19,517 between 2020 and 2025. Annual costs were projected at C$880.7 and C$703.1 million in 2025, for continuous and fixed OTT scenarios, respectively. Correspondingly, fixed OTT would provide a total cost reduction of C$213.8 million (5.94%) from 2020 to 2025, compared to continuous OTT. Conclusions: Fixed OTT is expected to result in major reductions in cost burden over the 5-year projection, compared to continuous OTT.</jats:p>	[ { "section_content": "Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia, representing 44% of all leukemia cases in Canada in 2016 [1].CLL is often diagnosed in the elderly, with a mean age at diagnosis of 71 years.In most patients, CLL is initially managed through watchful waiting (WW), with treatment initiation required after a median time of 4.8 years of surveillance [2].Although CLL treatments are non-curative, the recent development and availability of highly effective therapies has resulted in prolonged survivals. The selection of treatment regimens and prognosis are influenced by the fitness and age of the patients, as well as mutation status.For example, mutations at the immunoglobulin heavy chain variable (IGHV) region gene are associated with more durable responses to chemoimmunotherapy (CIT) treatments, whereas deletion at chromosome 17p or TP53 gene mutations [del(17p)] is associated with a poor prognosis and resistance to chemoimmunotherapy [3]. The development of oral targeted therapies (OTT) has led to substantial improvements in CLL treatments.Although CIT regimens were the standard of care for CLL patients for many years, continuous OTTs have emerged as the current standard of care for CLL in Canada.Ibrutinib, the first in class Bruton tyrosine kinase (BTK) inhibitor, was first reimbursed in 2015 for patients who had received at least one prior therapy and were considered inappropriate for treatment or re-treatment with a fludarabine-based regimen, and later reimbursed for treatment-naïve patients starting in 2016-2017 for patients considered inappropriate for fludarabine-based treatment due to high-risk disease such as patients with del(17p) and, in some provinces, for high-risk patients including those who are young and fit with unmutated IGHV status as of 2018 [4,5].A second generation BTK inhibitor, acalabrutinib, was recently approved in Canada in 2019 and is now reimbursed by all provinces for previously untreated CLL as well as for CLL patients who have received at least one prior therapy [6,7].These continuous OTTs have superior efficacy, easier administration, and reduced toxicity as compared to CIT [8].In the first-line CLL RESONATE-2 trial, ibrutinib showed significant improvement over chlorambucil in progression-free survival (PFS) in patients with and without IGHV and without del(17p) mutations, with a 5-year PFS of 70%, compared to 12% in those treated with the chemotherapy comparator, chlorambucil [9].Overall survival (OS) rate also increased with ibrutinib, irrespective of high-risk clinical or genomic features [9,10].The median duration of ibrutinib treatment was reported as 57.1 months in first-line treatment and 41.0 months in second-line treatment, with some patients receiving treatment for more than 5-6 years [9,10].Since these OTTs are administered continuously until disease progression or toxicity, and given their relatively high cost, continuous OTT represents a major economic burden on the Canadian healthcare system despite the improvements on survival and disease control for individual patients [11]. Venetoclax, marketed as a single agent in 2016, was recently approved by Health Canada and recommended for reimbursement by CADTH as part of two fixed-treatmentduration regimens: (1) in combination with rituximab (VR) for 24 months for the treatment of adult CLL patients who have received at least one prior therapy, and (2) in combination with obinutuzumab (VO) for 12 months for the treatment of patients with previously untreated CLL [12,13].The VR indication received reimbursement in 2020 in all Canadian provinces, while the VO indication received reimbursement starting in late 2021 and is now funded in all provinces [12,13].The efficacy of VO and VR demonstrated that after treatment cessation, the venetoclax-based treatment combination continues to significantly improve PFS compared with CIT [14][15][16].VO demonstrated a 3-year PFS of 81.9% versus 49.5% in the chlorambucil plus obinutuzumab group 2 years following treatment cessation [14][15][16].The introduction of venetoclax-based combinations administered as fixed-duration treatment offers targeted CLL treatment options that allow for better budget predictability and potential cost savings to the healthcare system.Thus, this study aims to estimate the cost burden of CLL in Canada with the introduction of fixed-treatment-duration OTT compared to current continuous OTT, while taking into account the future prevalence of the disease. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "", "section_name": "Materials and Methods", "section_num": "2." }, { "section_content": "A previously published state transition Markov model, by Lachaine et al., was adapted to assess the introduction of fixed-treatment-duration OTT.The Markov model structure includes four health states (WW, first-line treatment, relapsed/refractory treatment, and death), as detailed in Lachaine et al. [11]. ", "section_name": "Model Structure", "section_num": "2.1." }, { "section_content": "The patient characteristics and estimations made for the incidence and prevalence of CLL in the Canadian population have been previously described [11].Briefly, patients were defined by age (<65, 65-70 or >70 years old), phase of CLL treatment (WW, first-line or relapsed/refractory) and fitness level as well as del(17p) or IGHV mutation.Model input parameters were updated with the most recent data available, as shown in Table 1. Similar to Lachaine et al., a warm-up period was incorporated into the model to estimate the prevalent population living with CLL in 2020.The warm-up period was used to build the study population in 2020 with the appropriate proportion of patients at the different stages of their disease.The 2020 population was calculated by adding new incident cases over a 10-year period, from 2010 to 2020, into the model, stratified using clinical practices from this period.The annual incidence of CLL per year was calculated using data from Statistics Canada [17].The total number of incident cases was calculated by applying the annual rate of CLL per patient to the total Canadian population on a yearly basis.After this warm-up period, the model generates a population reflecting 2016 epidemiological data.Since the prevalence could be overestimated due to PFS and OS clinical trial data overestimation compared to clinical practice (exclusion of co-morbidities, better stratification, patient ages, etc.), model calibration was carried out using real-world data (prevalence at a defined time, drug utilization, etc.) along with the Excel Solver function [11].The model assumptions are presented in Table 1. ", "section_name": "Patient Characteristics", "section_num": "2.2." }, { "section_content": "Within the four health states (WW, first-line treatment, relapsed/refractory treatment, and death), patients could enter the model either in WW or in the first-line treatment.If patients failed to respond to first-line treatment, they entered the relapsed/refractory health state.After failure to respond to a second-line treatment, patients entered a sub-health state of relapsed/refractory, where their disease progressed but death has not yet occurred (palliative state).Patients in each of the health states could transition to death.Patients within the model cannot revert to previous health states. The probabilities of health state transitions were estimated based on PFS and OS from pivotal clinical trials and all-cause mortality rates (Table 2).Trials were selected based on the best and most recent evidence available, including phase III trials, for each treatment regimen.PFS was used to estimate the transition from first-line treatment to relapse as well as the progression from the relapse (second-line treatment) state.OS was used to determine the transition to death of relapse patients who progressed on second-line treatment.Allcause mortality rates were used to determine the transition to death from WW, first-line treatment and relapse (patients responding to treatment only) health states.As previously stated, note that all parameters were calibrated to obtain a population reflective of realworld epidemiological data.All probabilities were adjusted to fit model cycles of 28 days. ", "section_name": "Simulated Clinical Pathway", "section_num": "2.3." }, { "section_content": "The treatment algorithms for each CLL patient were defined by multiple factors such as the line of treatment, patient characteristics, mutations and year of treatment.Algorithms were based on the Alberta Clinical Guidelines and adapted based on the comments of clinical experts in CLL [45].The selected treatments are reimbursed by most Canadian provinces and their entry within the treatment pattern occurs at the time of first expected reimbursement in a Canadian province.Compassionate use is not considered within this model since the objective is to capture the economic burden of CLL from the public health care system perspective. For the prevalent population, treatment algorithms were simulated from 2010 to 2020, which reflects the evolution of the standard of care and other therapies as well as changes in clinical practice, with the entry of continuous OTT.For the incident population, the treatment algorithms were simulated from 2020 to 2025, which reflects the evolution of the standard of care and other therapies, as well as changes in clinical practice, with the entry of fixed OTT (Figure 1a).An additional clinical scenario was considered, where continuous OTT would remain the standard of care in order to evaluate the impact of the introduction of fixed OTT as an alternative treatment option (Figure 1b).continuous OTT would remain the standard of care in order to evaluate the impact of the introduction of fixed OTT as an alternative treatment option (Figure 1b). ", "section_name": "Treatment Algorithms", "section_num": "2.4." }, { "section_content": "To estimate the economic burden of CLL from a public healthcare perspective, only direct medical costs were considered, which includes costs associated with drug acquisition, follow-up/monitoring, adverse events (AEs) and palliative care (Table 1).Each cycle of treatment is 28 days.For drug acquisition costs, the unit cost of each treatment was obtained from IQVIA Delta PA (November 2020); similarly to the previously published model, treatment regimens were obtained from Cancer Care Ontario (CCO), and a body surface area of 1.89 m 2 and a weight of 76 kg was used [11,46,47].For continuous OTT, drug costs were accumulated until treatment discontinuation, either due to relapse or other clinical reasons.For fixed OTT, drug costs were accumulated until treatment completion or until treatment discontinuation, either due to relapse or other clinical reasons.A probability of discontinuation for each 28-day cycle was estimated at 0.70% and 1.40%, ", "section_name": "Cost Data", "section_num": "2.5." }, { "section_content": "To estimate the economic burden of CLL from a public healthcare perspective, only direct medical costs were considered, which includes costs associated with drug acquisition, follow-up/monitoring, adverse events (AEs) and palliative care (Table 1).Each cycle of treatment is 28 days.For drug acquisition costs, the unit cost of each treatment was obtained from IQVIA Delta PA (November 2020); similarly to the previously published model, treatment regimens were obtained from Cancer Care Ontario (CCO), and a body surface area of 1.89 m 2 and a weight of 76 kg was used [11,46,47].For continuous OTT, drug costs were accumulated until treatment discontinuation, either due to relapse or other clinical reasons.For fixed OTT, drug costs were accumulated until treatment completion or until treatment discontinuation, either due to relapse or other clinical reasons.A probability of discontinuation for each 28-day cycle was estimated at 0.70% and 1.40%, for first-line and relapse patients, respectively, as previously described in Lachaine et al. [11,20]. The follow-up and monitoring costs consisted of those involved in laboratory tests, as well as administration and professional fees for nurses, pharmacists, and physicians.Routine laboratory tests are needed for all CLL patients under treatment or WW.The unit costs of laboratory tests were retrieved from the Schedule of Benefits for Laboratory Services from the Ontario Ministry of Health and Long-Term Care [22], while the testing frequency was obtained from CCO and validated by clinical experts in CLL.The costs for administration comprised the cost of chemotherapy infusion as well as professional fees, including the hematologist, nurse, and a pharmacist cost.The professional fee for the hematologist was retrieved from the Schedule of Benefits-Physician Services in Ontario, while the nursing and pharmacy workloads were determined by CCO and Statistics Canada, and their median wages obtained from Job Bank Canada [23][24][25].The frequency of administrations per cycle was determined from CCO treatment regimens [46]. AEs were extracted from clinical trials and product monographs.The main AEs considered in the model include anemia, neutropenia, febrile neutropenia, thrombocytopenia, infection, and atrial fibrillation.Only grade 3 or 4 AEs were considered in this model.The cost per event was obtained from the Ontario Case Costing (OCC) analysis tool and the proportion of AE-managed inpatient or outpatient was determined by clinical experts in CLL [26]. Costs of palliative care were obtained from the Canadian Institute for Health Information (CIHI) patient cost estimator [48]. ", "section_name": "Cost Data", "section_num": "2.5." }, { "section_content": "The number of CLL patients as well as total costs associated with CLL management in Canada were projected from 2020 to 2025.The total annual costs for first-and secondline treatments were calculated for both continuous-and fixed-treatment-duration OTT.All costs were converted to 2020 Canadian dollars and presented in rounded values to simplify comprehension. ", "section_name": "Model Outcomes", "section_num": "2.6." }, { "section_content": "One-way sensitivity analyses (OWSA) were performed to assess the robustness of the model results.Since PFS, OS, and probability of discontinuation were directly varied through model calibration, they were not included in the sensitivity analyses.All other model parameters were varied with a range of ±25%. ", "section_name": "Sensitivity Analysis", "section_num": "2.7." }, { "section_content": "", "section_name": "Results", "section_num": "3." }, { "section_content": "As rates of incidence and survival are both increasing, the prevalence of CLL in Canada is projected to increase 1.3-fold from 15,512 patients in 2020 to 19,517 or 19,513 patients in fixed or continuous OTT scenarios, respectively (Figure 2a). ", "section_name": "Disease Burden", "section_num": "3.1." }, { "section_content": "", "section_name": "Cost Burden", "section_num": "3.2." }, { "section_content": "Under the continuous OTT scenario, the total annual cost of CLL management is projected to increase from C$352.5 million in 2020 to C$880.7 million in 2025, a 2.5-fold increase (Figure 2b).The steady increase in cost reflects the costs of continuous treatment duration OTT as well as the period between 2021 to 2025 when ibrutinib becomes available for all patients except for those with mutated IGHV in first-line treatment.In comparison, the fixed OTT scenario is projected to increase 2.0-fold from C$352.6 million to C$703.1 million during the same time period.Although initially, the addition of treatment with the combination of venetoclax and rituximab during 2020, as well as the combination of venetoclax and obinutuzumab starting from 2021, generated a sharper increase in cost than the continuous OTT scenario, the cost of fixed OTT starts to plateau in the year 2023, once patients complete their fixed-duration treatment.The total cumulative costs over the projected 5-year period were estimated at C$3.596 billion for the continuous OTT scenario and C$3.382 billion for the fixed OTT scenario.Consequently, the fixed OTT scenario represents a total cost reduction of C$213.8 million (5.94%) from 2020 to 2025, compared to the continuous OTT scenario. ", "section_name": "Total Annual Cost of CLL", "section_num": "3.2.1." }, { "section_content": "In first-line treatment, the cost of CLL treatment in the continuous OTT scenario is projected to increase from C$256.8 million to C$705.9 million between 2020 and 2025.In contrast, the cost in the fixed OTT scenario is projected to increase to C$536.5 million by 2025.The total costs in first-line treatment over the projected 5-year period was estimated at C$2.735 billion for the continuous OTT scenario and C$2.540 billion for the fixed OTT scenario.Consequently, the fixed OTT scenario represents savings of 7.15% (C$195.7 million) compared to the continuous OTT scenario. ", "section_name": "Cost of First-Line Therapy for CLL", "section_num": "3.2.2." }, { "section_content": "In second-line treatment, the cost of CLL treatment in the continuous OTT scenario is projected to increase from C$93.9 million to C$172.2 million from 2020 to 2025.In ", "section_name": "Cost of Second-Line Therapy for CLL", "section_num": "3.2.3." }, { "section_content": "In second-line treatment, the cost of CLL treatment in the continuous OTT scenario is projected to increase from C$93.9 million to C$172.2 million from 2020 to 2025.In contrast, the cost in the fixed OTT scenario is projected to increase to C$164.1 million in 2025.The costs of second-line treatment over the projected 5-year period was estimated at C$847.0 for the continuous OTT scenario and C$829.2 billion for the fixed OTT scenario.Thus, costs are decreased by 2.10% (C$17.8 million) under the fixed OTT scenario. ", "section_name": "Cost of Second-Line Therapy for CLL", "section_num": "3.2.3." }, { "section_content": "The OWSA showed that the cost of CLL management was most sensitive to the cost of ibrutinib, probability of watchful waiting at diagnosis, cost of venetoclax, cost of obinutuzumab, as well as neutropenia.The tornado diagram presenting the difference in total costs of CLL management from 2020 to 2025 between the fixed OTT and continuous OTT scenarios is presented in Figure 3. urr.Oncol.2023, 30 contrast, the cost in the fixed OTT scenario is projected to increase to C$164.1 million in 2025.The costs of second-line treatment over the projected 5-year period was estimated at C$847.0 for the continuous OTT scenario and C$829.2 billion for the fixed OTT scenario Thus, costs are decreased by 2.10% (C$17.8 million) under the fixed OTT scenario. ", "section_name": "Sensitivity Analysis", "section_num": "3.3." }, { "section_content": "The OWSA showed that the cost of CLL management was most sensitive to the cost of ibrutinib, probability of watchful waiting at diagnosis, cost of venetoclax, cost of obinutuzumab, as well as neutropenia.The tornado diagram presenting the difference in total costs of CLL management from 2020 to 2025 between the fixed OTT and continuous OTT scenarios is presented in Figure 3. ", "section_name": "Sensitivity Analysis", "section_num": "3.3." }, { "section_content": "This study provides the first estimate of the economic burden related to CLL management with the launch of fixed-treatment-duration OTT in Canada.Although continuous OTT is currently the standard of care for CLL patients, fixed OTT, which was recently introduced to the Canadian healthcare system, has an added benefit of considerably re- ", "section_name": "Discussion", "section_num": "4." }, { "section_content": "This study provides the first estimate of the economic burden related to CLL management with the launch of fixed-treatment-duration OTT in Canada.Although continuous OTT is currently the standard of care for CLL patients, fixed OTT, which was recently introduced to the Canadian healthcare system, has an added benefit of considerably reducing the cost burden for the treatment of CLL patients over the 5-year projected period.Our study estimates that from 2020 to 2025, there will be a 1.3-fold increase in the number of people living with CLL because of the increased disease incidence as well as improved survival under OTT treatment.Concurrently, the overall cost of CLL management in the continuous OTT scenario is predicted to increase to C$880.7 million by 2025, a 2.5-fold increase from 2020.The overall cost is predicted to reduce to C$703.1 million with the availability and adoption of fixed-treatment-duration OTT.It is however unknown how retreatment with finite OTT will impact the costs as the studies did allow for retreatment, and how the costs would be combined with continuous OTT once patients no longer respond to finite treatments.In the meantime, in alignment with patient values for tolerable, safe treatment options, implementation of fixed OTT could alleviate the financial burden associated with CLL treatment. In a previously published study comparing the cost burden of continuous OTT to traditional CIT treatment, the annual cost of CLL management in 2025 was estimated to increase to C$957.5 million with the adoption of continuous OTT, representing a 15.7-fold increase in costs compared to the CIT scenario.This current study extends the comparison between continuous OTT and fixed-treatment-duration OTT, with total costs of continuous OTT in 2025 estimated at C$880.7 million.For this current study, most model parameters from our previous publication have remained similar, except for the inclusion of new treatments, recent clinical trial data and cost updates.In line with these modifications, results for continuous OTT are within the same price range and thus, demonstrate the robustness of the model. Although the economic burden of fixed OTT in CLL has not been otherwise conducted, budgetary impact analyses (BIA) have been performed for VO and VR for a fixed treatment duration of 12 and 24 months, respectively, in the United States (US) and France [49][50][51].In the US, the implementation of VO results in a cost saving of US$1.6 million per 1 million members under the US health plan over a 3-year time horizon [50].Similarly, VR resulted in a cost saving of US $0.7 million [49].In France, a BIA analysis suggested that although there is an increase in total cost when the combination of VO is implemented during year 1, it is followed by a cost saving in years 2 through 10, compared to other CLL treatments.Over the ten-year time horizon, the fixed-duration treatments allowed a total cost saving of EUR $860 million [51].Therefore, although these studies are not directly comparable to our economic burden study, the conclusion is consistent that implementing fixed-treatment-duration OTT can potentially alleviate economic burden on the healthcare system. Our analysis has several limitations.First, patients receiving treatment in the model can only receive first-or second-line treatment.However, this does not necessarily reflect the clinical setting, as patients can also receive third and subsequent-line treatments.Although this option is excluded in our analysis, the costs included in this study reflect our objective of comparing the cost of continuous versus fixed OTT.Additionally, a fixed rate of CLL patients with del(17p) mutation was assumed for both first-and second-line treatments.However, the percentage of patients with del(17p) mutation can increase to more than 30% over time, particularly in relapsed patients [52].Furthermore, the hospitalization due to dose ramp up of venetoclax was not considered in our model, though other cost factors of laboratory testing and nursing/physician assessments were included.However, according to the DEVOTE study, an observational study of relapsed/refractory CLL patients initiating VR or venetoclax in routine clinical practice in Canada, the utilization of hospitalization due to dose ramp up varies greatly across Canada [53].According to study results, approximately one third of patients were hospitalized at venetoclax initiation (median duration of 2 days), and venetoclax hospitalizations for subsequent dose ramp ups were even less frequent.Therefore, the exclusion of this cost parameter is not expected to greatly impact the results of the study.The use of obinutuzumab in combination with venetoclax may increase costs due to the nature of the infusion and the time it takes to deliver it.There may also be associated drug costs and hospitalizations that are not accounted for. Lastly, to manage toxicities and adverse events associated with OTT, dose reduction could be required for patients on ibrutinib and venetoclax [54][55][56][57].As data supporting the clinical efficacy of dose reduction are largely from real world experience, they are observational and need to be formally studied to establish definitive clinical practice guidelines [56,58,59].Therefore, dose reductions for either fixed or continuous OTT were not considered in this study.However, it is possible that use of lower doses in clinical practice due to AE management can lower the economic burden in both continuous and fixed OTT scenarios in the future.To partially assess this limitation, a sensitivity analysis varying the unit cost of ibrutinib and venetoclax by ±25% was performed; both upper and lower bound results showed a reduction in the economic burden.In addition, it is also possible that the rates of discontinuation are overestimated as they were derived based on the rates of previous chemotherapies.In the literature, the rates of discontinuation of OTT range between 15-20%; however this data is still emerging [20]. ", "section_name": "Discussion", "section_num": "4." }, { "section_content": "In conclusion, this study highlights that although prevalence and cost associated with CLL in Canada are predicted to increase, the introduction of fixed-treatment-duration OTT will lead to a reduction in the cost burden of CLL treatment.Changes in pricing and clinical practices, such as dose reduction for adverse event management or more targeted and personalized treatment regimens, may further help alleviate the economic burden of CLL management. ", "section_name": "Conclusions", "section_num": "5." } ]	[ { "section_content": "Acknowledgments: PeriPharm Inc. would like to thank Maggie Dong for her assistance in writing the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "Funding: This research received external funding from AbbVie Canada.No grant number applicable. Informed Consent Statement: Not applicable. ", "section_name": "Institutional Review Board Statement: Not applicable.", "section_num": null }, { "section_content": "Funding: This research received external funding from AbbVie Canada.No grant number applicable. ", "section_name": "", "section_num": "" }, { "section_content": "Informed Consent Statement: Not applicable. ", "section_name": "Institutional Review Board Statement: Not applicable.", "section_num": null }, { "section_content": "Code L223, 222, 191, 029, 030, 031, 005, 208, 700 [22] CBC panel 14.74 * Code L393, 700 [22] Coagulation parameters 13.42 * Code L445, 700 [22] Serology 21.01 * Code L319, 700 [22] Chemotherapy infusion, administration, and management 105.15Schedule of benefits.Code G359 [23] Data Availability Statement: The data presented in this study are available within this article. ", "section_name": "", "section_num": "" }, { "section_content": "Author Contributions: The guarantor of the article is J.L., K.G. was involved in study conception and design, modelling, analysis and interpretation of data, and drafting the manuscript.J.L. was involved in study conception and design, revision of the manuscript, and study supervision.A.A., V.B., I.F. and C.O. were involved in study conception and design and revision of the manuscript.All authors have read and agreed to the published version of the manuscript. Conflicts of Interest: J.L. is a partner at PeriPharm Inc., a company that has served as a consultant to AbbVie and has received funding from AbbVie.J.L. and K.G. from PeriPharm Inc., have participated in the study conduct, data interpretation and the preparation of the manuscript.A.A. has received honoraria from Abbvie and Astra Zeneca accepted into a separate account within the Ottawa Hospital Research Institute, for research/academic use only.V.B. has received research funding from CIHR, CancerCare Manitoba, Research Manitoba, Janssen and Abbvie and has served as a consultant to Abbvie, Janssen AstraZeneca, Gilead, Roche, and Lundbeck.I.F. has provided advisory consultations for Abbvie, AstraZeneca, BMS, Gilead, Janssen, Merck, Novartis, Roche and Seattle Genetics and has given presentations for Abbvie, Janssen, Novartis, Roche.C.O. has received honoraria from AbbVie, Astrazeneca, Janssen, Roche, Merck, Servier, Incyte.No author has received funding for developing the manuscript.AbbVie participated in the design and provided financial support for the study.AbbVie reviewed and approved this publication. ", "section_name": "", "section_num": "" } ]
10.1371/journal.pone.0039808	Crowd Sourcing a New Paradigm for Interactome Driven Drug Target Identification in Mycobacterium tuberculosis	Una década después de la disponibilidad de la secuencia del genoma de Mycobacterium tuberculosis (Mtb), ningún fármaco prometedor ha visto la luz del día. Esto no solo indica los desafíos para descubrir nuevos medicamentos, sino que también sugiere una brecha en nuestra comprensión actual de la biología de Mtb. Intentamos cerrar esta brecha llevando a cabo una amplia re-anotación y construyendo un mapa de interacción de proteínas a nivel de sistemas de Mtb con el objetivo de encontrar nuevos candidatos a dianas farmacológicas. Hacia esto, sinergizamos el crowdsourcing y los métodos de redes sociales a través de una iniciativa 'Connect to Decode' (C2D) para generar el primer y más grande interactoma curado manualmente de Mtb denominado 'vía del interactoma' (IPW), que abarca un total de 1434 proteínas conectadas a través de 2575 relaciones funcionales. Se han catalogado las interacciones que conducen a la regulación génica, la transducción de señales, el metabolismo y la formación de complejos estructurales. En el proceso, hemos anotado funcionalmente el 87% del genoma de Mtb en el contexto de los productos génicos. Además, combinamos IPW con UNA red basada en CADENAS para informar proteínas centrales, que pueden evaluarse como posibles dianas farmacológicas para el desarrollo de fármacos con los menores efectos secundarios posibles. El hecho de que cinco de las 17 dianas farmacológicas previstas ya estén validadas experimentalmente, ya sea genética o bioquímicamente, da crédito a nuestro enfoque único.	[ { "section_content": "Proclaimed a global health emergency by the World Health Organization (WHO) in 1993, Tuberculosis (TB) still remains the leading cause of mortality and affects approximately 32% of the world population [1].The emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the vulnerability of the patients infected with HIV to tuberculosis have not only fuelled the spread of the disease but also present a challenging task of understanding the disease physiology and discovering new drug targets.In this quest, Mtb was sequenced and annotated in 1998 [2].A subsequent re-annotation in 2002 successfully assigned functions to almost half of the approximately 4000 genes [3].More recently, 20 more ORFs have been added to this list and the annotations updated [4,5].However a huge gap in information exists between published literature and the genome databases.The existing annotations in these databases are thus insufficient to generate the protein interaction map or the interactome, pivotal to understanding Mtb biology and identification of novel drug targets.To this end, Open Source Drug Discovery (OSDD) project (www.osdd.net)[6,7] launched the Connect to Decode (C2D) program (http://c2d.osdd.net),an innovative blend of crowd sourcing and social networking in a virtual cloud space for a comprehensive collaborative reannotation of Mtb which is the primer for generating the interactome.The ultimate objective is to identify drug targets based on better understanding of the complex interactions of various biological macromolecules in the pathogen. Systems biology-based approaches have been applied to obtain better insights into the pathogen biology [8].This strategy may help in identifying more than one potential drug targets and these can be utilized as sets of targets for a polypharmacology approach.A promising candidate in this category is bi-substrate acylsulfamoyl analogues that simultaneously disrupt crucial nodes in biosynthetic network of virulent lipid with dramatic effect on the cell surface architecture of Mtb [9].Also, a recent study on genome-wide siRNA experiment has identified host factors that regulate Mtb load in human macrophages and are crucial to understand the dynamic interplay of molecular components of the pathogen and the host [10].There are many such studies that try to capture the snapshots of the molecular interactions in Mtb in different conditions.It is therefore imperative to capture and curate data on experimentally validated interactions lying scattered in diverse sources in the literature to generate a genome scale network.This was achieved through the C2D program.The C2D community started with initial registration of more than 800 researchers, which largely consisted of research scholars, graduate students and under-graduate students.The participants were trained, evaluated and filtered at various stages of online training and assignments (https://sites.google.com/a/osdd.net/c2d-01/pathwayannotationproject/results-of-the-exercise).More than 100 researchers were selected as curators to obtain the final annotations (https://sites.google.com/a/osdd.net/c2d-01/pathwayannotationproject). Here we describe how C2D has implemented a community annotation approach in a distributed co-creation mode for mining literature and how the accuracies and scope of assigning functions were enhanced using combined evidence approach.We have enriched the annotations of the Mtb genome both in terms of coverage and details (Table 1).Web2.0 collaborative online tools enabled voluntary community participation for implementing this task.An important part of the project was creating self-organized communities to collectively learn and share the process and the standards for reporting annotations.As per published estimates, this innovative approach packed nearly 300 man-years into 4 months [11] and it has also established a novel way of collective problem solving on a voluntary basis in a sustainable manner [12].This is, to the best of our knowledge, lead to the creation of the largest manually curated interactome of Mtb.Based on the varied nature of interactions among proteins in vivo, we propose a new network definition called ''Protein-Protein Functional Network'' (PPFN).This network encompasses a total of 1434 proteins connected through 2575 functional relationships.In this paper, we detail how the Interactome -PathWay (IPW), an open collaborative platform was used to generate and analyze potential drug targets.Using betweenness centrality [13] as a first indicator to shortlist candidate drug targets, we zeroed into 73 proteins.We have in the process also created a sustainable open innovation platform. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Results and Discussion", "section_num": null }, { "section_content": "An overview of the approach followed in 'Connect to Decode' (C2D) exercise is as illustrated in Figure 1.Broadly the approach was designed based on the principles of the fourth paradigm of science, encompassing data collation, curation and analysis [14].Roughly ,4.4 Mbp genome of Mtb was re-annotated manually.To streamline the annotation process and select a community of researchers competent to implement this project, a series of online assignments and training modules were assigned (see methods).These steps ensured the selection of serious and dedicated contributors thereby assuring the quality of data collation, curation and analysis.Various standard operating protocols (SOPs) were designed and shared with the participants for the consistency in the steps followed for the annotation of genes (https://sites.google.com/a/osdd.net/c2d-01/pathwayannotationproject/instructionsfor-annotation and https://sites.google.com/a/osdd.net/c2d-01/pathwayannotationproject/example-annotation and https://sites.google.com/a/osdd.net/c2d-01/pathwayannotationproject/stepsforproteinannotation ).Given the exponential increase in the number of publications from about 300 per year since 1990's to a staggering 2000 per year in 2010, the challenging task of collating and curating data was achieved through the formulation of community editable interactive platform designed to facilitate real time annotations and continuous updates.The community scanned and retrieved information from nearly 10,000 published studies in addition to extracting information from databases and transferred annotations using sequence and structure analyses based approaches.The community has cited more than 3000 papers in annotation process as on an average 3-4 manuscripts were referred or read in order to get the relevant information to annotate a given protein. ", "section_name": "C2D Annotation", "section_num": null }, { "section_content": "IPW has resulted in annotation of 87% of the genome in the context of reporting gene products as compared to 52% in the reannotation reported in 2002.Moreover, less than 5% of the interactions in IPW (Table S1) exist in other manually curated interaction databases such as BIND [15], APID [16], IntAct [17], DIP [18] and MINT [19] (Figure 2(b)).Thus, to the best of our knowledge, Connect to Decode's Interactome Pathway Annotation (IPW) has generated the largest data set of manually curated interactions in Mtb.These interactions not only include data from large interaction databases such as IntAct, BIND, MINT, APID, Table 1.The data structure that was used to capture the interactome data.DIP, etc but also include a large amount of manually curated information from literature. Of the 1193 hypothetical proteins from TubercuList [4], the IPW based annotations identify gene products for 770 proteins.Of the 1480 hypothetical proteins reported in KEGG [20] database, functional associations have been made to 1055 proteins, clearly showing how IPW has bridged the wide gap that existed between information captured in databases and that available in literature.To ensure that IPW remains up to date, the data from IPW is shared with members of the OSDD community in an 'edit' mode, through which new interactions can be added using the SOP that includes a rigorous quality check phase, specifically designed for community contribution. ", "section_name": "The Mtb Genome Annotation and Interactome Curation", "section_num": null }, { "section_content": "Interactome as a whole constitutes various biological interactions belonging to both structural and functional type of proteinprotein associations.To have an encyclopedic view of various interactions that take place at protein functional level, we report the construction of two types of networks.The first network, termed IPW only (Figure 2(a)), was constructed on the basis of the IPW curated data alone.The nodes in the network represent the proteins whereas the edges represent the functional interactions among those proteins.The nodes were scaled and color coded in proportion to their degrees.Also, based on the common interactions we derived a connectivity relationship between various TubercuList functional classes [4].Figure 2(c) shows the connectivity among 10 broad functional classes of Tubercu-List.The edge thickness was taken to be directly proportional to the number of common proteins between the two TubercuList functional classes for the given pair.Significant functional dependencies are seen among the 'Lipid Metabolism, Cell Wall, Intermediary metabolism and Regulatory systems' functional classes, reflected in their edge thicknesses in the network.Disruption of such linkages can lead to breakdown of crosstalk between these biological processes and thus could be exploited to identify new drug targets. Secondly, in order to obtain insights on the complete functional organization among all the possible proteins of Mtb, a combined network termed, IPW-STRING (IPWSI), was constructed by overlaying STRING network on the IPW network.The STRING based network of Mtb was derived from STRING 8.0 [21] database consisting of various interactions among proteins as derived on the basis of extensive computational and limited experimentally inferred interactions.Computational predictions have been based on established methods such as phylogenetic profiling, domain fusion, common gene neighborhood and operon criteria.However, computational models over predicts interactions since they do not account for spatio-temporal separation of the interacting partners.Thus, in the combined network the accuracy of interaction decreases whereas the coverage increases.It should also be noted that there is an inherent bias for well-studied proteins in IPW.A simple comparison shows that nearly 60% of IPW interactions have experimental evidence codes as compared to 2% existing in STRING.Also, about 29 additional proteins and 1762 new functional interactions apart from that reported by STRING were included in the new IPW-STRING combined interactome. The combined IPW-STRING interactome was further used to decipher various possible drug targets using the concepts of graph theory.The network analysis of these networks provides a means to understand the functional organization of the organism from the network topology point of view [22,23].Various network properties as computed for both the networks and their biological relevance are discussed below. ", "section_name": "Interactome Construction: IPW and Combined Network with STRING", "section_num": null }, { "section_content": "In order to understand the functional organization of constructed interactome we further assessed the fundamental properties of this network from the graph theoretic point of view.Given a vast interaction space encompassing the interactome as whole, where the nodes represents proteins and interaction represents a functional relation between them, it becomes imperative to understand the functional organization of the network from its topology.The most fundamental characteristic of a graph is the connectivity of its constituent nodes as represented by the degree.Degree, being a measure of interconnectedness of nodes highlight the importance of a node (protein in this case) with respected to other nodes in the network.A maximum degree of 44 and 289 was observed for the IPW and IPWSI networks, respectively, suggesting the level of maximum number of functional relation of a given protein in both the networks. Clustering coefficient for a node indicates the connectivity of the neighbours of a given node to the other nodes in the network [24].This parameter was computed to elucidate the dependencies of two or more proteins with respect to each other and to rest of the proteins in the network.The clustering coefficient for IPW and IPWSI networks was observed to be 0.249 and 0.377, respectively.The high clustering coefficient of both the networks suggests the presence of well-connected hubs within the network, which are important from the functional crosstalk between the proteins of Mtb.Further, the characteristic path length of both the networks was computed in order to comprehend the extent of functional relation between any two given proteins in the network.The characteristic path length of both the networks is as shown in Figure 3(a).The characteristic path length in IPW network was observed to be 7.2 whereas for IPWSI it was observed to be 3.13.From the network navigability point of view the characteristic path length can be inferred as the number of steps that one has to take traversing from one node to other, which from biological point of view could be inferred as the amount of communication that is possible between any two proteins.Pertaining to the high characteristic path length of IPW alone, the absence of functional relation between any two proteins can be inferred; however, the functional relation between any two proteins increase when the IPW alone was clubbed with STRING based network.The characteristic path length, thus, can be used to understand the functional gap that possibly exists in the protein-protein interaction network.Emphasizing on the network communication further, the network diameter was computed representing the length of the 'longest' shortest path in the network.The network diameter of IPW and IPWSI networks was observed to be 18 and 10, respectively.Akin to characteristic path length, the network diameter can be used to interpret the overall navigability of the network, the higher the diameter, the more distantly two nodes are related and vice versa. As discussed, understanding the topological organization of the network could lead to better understanding of its underlying principles.The network topology could also be used to understand the number of possible modules (hubs) in the network, which may help in identifying potential drug targets.In order to obtain such insights, we tested the existence of power law distribution on IPW and IPWSI networks, respectively.The power law distribution can also be used to understand the scale free nature of a network [23].There is extensive literature that reports the existence of scale free nature of biological networks.The power law distribution on the node degree distribution of IPW and IPWSI networks is shown in Figure 3(b).The value of c was observed to be 1.99 for IPW and 2.01 for IPW-STRING combined node degree distribution. ", "section_name": "Topological Organization of Interactome", "section_num": null }, { "section_content": "Apart from inferring fundamental principles of network properties the availability of an interactome also enables prediction of essential proteins from the network structure point of view.The protein lethality within a network is usually obtained from the degree distribution of the nodes in the networks.The nodes with high degree are considered important and hence regarded as probable drug targets.The degree distribution alone could lead to improper putative drug target identification as it does not capture the alternate routes in the network.Most of the biological networks possess large number of shortest paths [25].The large number of shortest paths also suggests the availability of alternate routes within the network which could be used to achieve a certain biological objective.Removing such nodes from the network could lead to maximum disruption in the network.In order to capture these properties, important nodes as well as important edges, we used betweenness centrality [24,26] as a metric system to infer putative drug targets.The node betweenness centrality at a threshold of $0.2 lead to identification of 17 and 64 central proteins from IPW and IPWSI networks, respectively (Table S2). ", "section_name": "Target Identification", "section_num": null }, { "section_content": "To design a viable drug it is essential to ensure least probability of off-target interactions.A sequence, structure and systems based analysis was performed in order to predict the druggability of the shortlisted central proteins from the two networks so as to reduce the chances of off-target interactions. The list of 17 and 64 proteins (73 unique proteins as eight are common in the two lists) was first filtered against human homologs and human oral and gut flora [27].Of the 17 targets identified by IPW, none had a homolog in human proteome and in human oral and gut flora.In the combined network IPWSI, 53 such targets were identified (Figure 4).There are 62 unique central proteins without any significant homology to human proteome, gut and oral flora from IPW and IPWSI.We further analyzed this list of 62 proteins for absence of small peptides (octamer) since it has been reported that a small fraction of peptide sequences are evolutionarily conserved and invariant across several organisms [28].These peptide sequences can adopt similar conformation in different protein structures [28].A comparative analysis shows that one protein Rv3221A does not share any common octapeptide with human proteome, gut or oral flora.However, a closer and detailed analysis needs to be performed for proteins sharing octapeptide with human proteome and human microbiome in order to evaluate their status for off-target binding.In order to understand the binding pockets, an independent analysis has been performed to predict and match binding pockets of central proteins with human proteome.Of the 73 central proteins, 57 have either PDB or ModBase [29] structure making them amenable to structural analysis for druggability.We analyzed these proteins as per the targetTB [30] pipeline where the top 10 binding sites for each of these 57 proteins were identified using PocketDepth algorithm [31].The binding pockets of these proteins were then compared with human proteome using PocketMatch [32].Of the 57 proteins, 31 proteins have high structural similarity with human proteome at binding site level whereas 26 proteins which do not have binding site similarity with human proteome.It is interesting to note that seven of these are experimentally validated drug targets. Rv3221A (RshA) (Figure 4 List d), an anti-sigma factor to the primary stress response sigma factor SigH, passed all filters but is neither reported as a potential drug target in literature nor in targetTB predictions.The gene encoding RshA lies in the same operon as SigH and is co-expressed with the same [33].It has a strong affinity to bind with SigH and attenuates its ability to bind to the RNA polymerase holoenzyme under normal growth conditions.Under conditions of oxidative stress, phosphorylation of RshA by Rv0014c (PknB) abolishes its binding to SigH, which in turn leads to the cascade of expression of several stress response proteins [34] (Figure 5).SigH causes increased expression of two other sigma factors Rv2710 (SigB) and Rv1221 (SigE), which are also known to be stress related sigma factors that assist Mtb in its survival during several stress conditions and are also central proteins.The other interacting partners of RshA include heat shock proteins and chaperones like Rv0384c (ClpB) and Rv0350 (DnaK), enzymes for oxidative stress response Rv1471 (TrxB1), Rv3913 (TrxB2) and Rv3914 (TrxC) which are also part of the sigH regulon.sigH also induces enzymes involved in cysteine biosynthesis and in the metabolism of ribose and glucose for the production of mycothiol precursors, which assist in cellular protection under oxidative stress.The SigB and SigE signaling cascade downstream interacts and regulate other central proteins (Figure 5).RshA is at the beginning of this cascade and seems to play in crucial role in regulating the stress response proteins, starting with sigH. The objective of the interactome construction and analyses was to identify central proteins, which have significant roles in maintaining growth and survival of the bacterial pathogen.We identified 17 such central proteins (Table 2), five of which (PknB, NuoG, PhoP, EccCb1, HspX) have been previously functionally characterized and shown to be essential by gene deletion and mutation and thus are considered as validated drug targets.The target gets further validated if there are inhibitors which inhibit the function of the target enzyme or protein as well.PknB (Rv0014c) is an essential serine-threonine protein kinase in Mtb and has role in a number of signaling pathways in cell division and metabolism.Several inhibitors have been reported for this kinase [35] and is also one of the targets being pursued by Working Group on New TB Drugs (http://www.newtbdrugs.org/project.php?id = 81).NuoG (Rv3151) is a subunit of type I NADH dehydrogenase playing an important role in growth in macrophage and pathogenesis in animal models [36].PhoP (Rv0757), a response regulator and part of the two component system, when mutated leads to growth defects in macrophages and in mouse models [37].eccCb1 (Rv3871) is a part of the RD1 S2).doi:10.1371/journal.pone.0039808.g004region and mutation leads to attenuated growth and toxicity in THP-1 cells.The mutants cannot export CFP-10 and are avirulent [38].hspX (Rv2031c) encodes for a alpha-crystallin-like protein and plays a significant role in retaining a non-replicating state in latency [39,40].The fact that five of the 17 putative drug targets from IPW are already validated drug targets, lends credence to our approach of annotating the genome and interactome construction of Mtb for system level understanding towards novel drug target identification. Despite the efforts over a number of years, discovering novel, fast acting drugs for TB has been a major challenge.However, recently introduced combination drug Risorine designed on the principles of Ayurveda has been shown to cut down rifampicin use leading to very high compliance [41].Understanding the biology of the pathogen through a systems level approach can help in identifying the Achilles heel for Mtb.Towards this, Interactome Pathway annotation has captured the updated relevant information on Mtb genes and has tried to unravel the puzzle.We have amalgamated crowd sourcing with social networking to comprehensively reannotate the Mtb genome, generated its largest ever interactome and propose potentially efficacious drug targets.In the process, we have set up an open collaborative platform and a dynamic community to ensure regular updates. ", "section_name": "Analysis of Putative Drug Targets: Identifying Probable Non-toxic Targets", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "Data capture.Two annotation standard operating protocols (SOPs), in the presence of literature and in the absence of literature, were designed in order to capture the maximum amount of relevant data.Wherever the protein was not studied in Mtb, the annotations were transferred from other organisms based on conservative statistical measures in sequence and structurebased analysis as discussed below (i and ii).To ensure consistency and integrity of the data added to the resource, Standard Operating Protocols (SOPs) were created and followed by the community.These SOPs and tutorials may be accessed at (http:// c2d.osdd.netand https://sites.google.com/a/osdd.net/c2d-01/pathwayannotationproject). Annotation SOP in presence of literature.The first step was to retrieve information on Mtb proteins with experimental evidence from literature.PubMed and Google based literature searches were carried out using suitable keywords, such as the respective Rv number, gene name, Mycobacterium tuberculosis, along with suitable Boolean expressions, such as AND and OR (for example, ", "section_name": "Crowd Sourcing for Data Curation", "section_num": null }, { "section_content": "Figure 5. Illustration of 17 putative drug target interaction from IPW interactome depicting the cascade of how the central proteins interact with each other in a spatio-temporal manner under different conditions like growth, stress and survival in macrophages including virulence.Under normal conditions, PknB phosphorylates RshA which inhibits SigH.However, under oxidative stress, RshA is not phosphorylated and this abolishes its binding to SigH, rendering it free.SigH in turn upregulates expression of SigE and SigB which regulates MprA (bacterial persistence regulator).MprA also regulates SigB and SigE.SigB plays important role in adaptation to stationary phase and nutritionally poor conditions and SigE is upregulated in mycobacterial growth within human macrophages and is transcribed from three different promoters under different conditions.sigB is also regulated by SigF, which regulates the expression of genes involved in the biosynthesis and structure of the mycobacterial cell envelope, including complex polysaccharides and lipids, particularly virulence-related sulfolipids and several transcription factors.Rv0516c is an anti-anti sigma factor and regulates anti-sigma factor SigF (upregulated during infection culture of human macrophages and in nutrient starvation condition; regulates transcription of genes involved in cell wall biosynthesis, sulfolipid metabolism, nucleotide metabolism, energy metabolism and several transcription factors) on getting phosphorylated by PknD which in turn is regulated by Rv0020c phosphorylated by PknB and PknE.SigF also regulates sigC and regulates hspx that is also regulated by dosR regulon.dosR regulon in turn is again regulated by PhoP which is a transcription factor for nuoG, eccCb1, esxb/cfp10 [48].doi:10.1371/journal.pone.0039808.g005manually scanning the available literature, emphasis was placed on the references, which dealt with Mycobacterium tuberculosis H37Rv followed, by evidence in other mycobacterial species.If the protein was an enzyme, the corresponding reaction, along with the EC number, and the pathway(s) in which the protein participates was also recorded. SOP for annotation in the absence of direct information from literature.In absence of direct literature information, annotations were derived based on sequence, structure and profile based information and analyses.To begin with, NCBI-BLAST [42] was used to obtain homology information of the query protein.Hits with e-value of #0.0001 and identity of $35%, with $75% sequence coverage were considered as significant hits.Annotations of the closest homologue were transferred and recorded in the template against each annotation.Similarity search in the Pfam [43] database was carried out to support BLAST results and also to annotate in the absence of high query coverage with BLAST analysis.If both BLAST and Pfam similarity search failed to give a significant hit, Phyre [44], an automatic fold recognition tool was used for predicting the function of the Mtb proteins through high confidence fold associations.Appropriate evidence codes have been used to distinguish between transferred annotations and experimental based annotations. Data curation.Multiple rounds of collaborative data quality checks were carried out to ensure that the data has been correctly captured and reported.A set of instructions (SOPs) were devised for the same (https://sites.google.com/a/osdd.net/c2d-01/pathwayannotationproject/data-qc-guide) where the annotations curated by the members were systematically crosschecked iteratively by other members.It was interesting to note that high quality curation was achieved by this approach of 'many eyeballs make the bug shallow', a common phenomenon in open source software projects. Data organization.The collated data was organized into a defined data structure as depicted in Table 1 with two columns, field and description.The PSI MI (Proteomics Standards Initiative Molecular Interactions) was used as the community standard for reporting protein-protein interactions in MITAB format (Table S3).This helps in improving the representation of molecular interaction data and its accessibility to the user community. ", "section_name": "[Rv1018c] AND [mycobacterium tuberculosis], [epoxide hydrolase] AND [mycobacterium tuberculosis]). While", "section_num": null }, { "section_content": "IPW and IPWSI network.The IPW-only network was constructed based on the annotations and curation of the data from IPW.The combined network of IPW and STRING termed, IPWSI, was constructed by combining the IPW network with that from STRING.All the interactions in STRING with high and medium level confidence score (above 400) were used to construct STRING based protein-protein interaction network.Methods used to compute network parameters are discussed below. Network properties.To understand the functional organization of interacting proteins in both the networks, an analysis of various network topologies was performed.These network properties were computed using Boost Graph library in MATLAB (David Gleich; http://www.stanford.edu/,dgleich/programs/matlab_bgl/). Connectivity or degree.The most elementary characteristic of a node in the network is its degree k, which represents, for a given node the number of other nodes it is connected to. Clustering coefficient.The clustering coefficient was first defined by Watts and Strogatz [24].The clustering coefficient, C, for a node is a notion of how connected the neighbours of a given node are to the other nodes (cliquishness) [45].The average clustering coefficient for all nodes in a network is taken to be the network clustering coefficient.In an undirected graph, if a vertex vi has ki neighbors, k i (k i -1)/2 edges could exist among the vertices Table 2.The list of all the 17 central proteins as predicted from the betweenness centrality of .0.2 through IPW network with their gene products. ", "section_name": "Interactome Construction and Network Parameter Estimation", "section_num": null }, { "section_content": "Gene Name Description (Gene Product) Rv0014c* [35] pknB within the neighbourhood (Ni ).The clustering coefficient for an undirected graph G(V, E) (where V represents the set of vertices in the graph G and E represents the set of edges) can then be defined as The average clustering coefficient characterizes the overall tendency of nodes to form clusters or groups.C(k) is defined as the average clustering coefficient for all nodes with k links. Characteristic path length.The characteristic path length, L, is defined as the number of edges in the shortest path between two vertices, averaged over all pairs of vertices.It measures the typical separation between two vertices in the network.Intuitively, it represents the network's overall navigability [45]. Network diameter.The network diameter d is the greatest distance (shortest path, or geodesic path) between any two nodes in a network.It can also be viewed as the length of the 'longest' shortest path in the network. where d G (u, v) is the shortest path between u and v in G [45]. Power law distribution.For a given network the power law distribution states the probability that a given node has k links, which is given by equation p(k) , k-c, where c is degree exponent.For smaller values of c, the role of the 'hubs', or highly connected nodes, in the network becomes more important.For c .3, hubs are not relevant, while for 2,c ,3, there is a hierarchy of hubs, with the most connected hub being in contact with a small fraction of all nodes.Scale-free networks have a high degree of robustness against random node failures, although they are sensitive to the failure of [23].The probability that a node is highly connected is statistically more significant than in a random graph [45]. Betweenness centrality.The betweenness centrality is the measure of vertex within a graph.For a given graph G(V,E), with n vertices, the betweenness CB (v) of a vertex v is defined as. where s st is the number of shortest path from s to t, and s st (v) is the number of shortest paths from s to t that passes from vertex v.The betweenness centrality analysis was performed for both the networks [45][46]. ", "section_name": "Accession", "section_num": null }, { "section_content": "Sequence homology with human proteome, oral and gut flora.The complete human proteome was downloaded from NCBI and BLAST was used to filter out the proteins, which had homology of greater than 45% with human protein.Human gut and oral flora constitutes the microbes that are considered to influence the physiology, nutrition, immunity and development of host.The complete proteome of 8-gut flora and 27 oral floras were downloaded.CD-HIT with similarity of 60% and a word size of 4 was used to compare the predicted proteins [27]. Binding site similarity with human proteome, oral and gut flora.We analyzed these proteins as reported in targetTB [30] pipeline where the top 10 binding sites for each protein was identified using PocketDepth algorithm [31].The binding pockets of these proteins were then compared with human proteome using PocketMatch [32]. Peptide level conformation comparison with human proteome, oral and gut flora.We analyzed the proteins for absence of small peptides (octamer) [28] across human proteome, gut or oral flora using in house PERL scripts. Literature based target validation.The predicted targets were further validated based on presence of existing functional evidence in literature.Data-mining and manual curation was performed to identify and document validated drug targets in Mtb.In addition to this, it was also documented whether the central protein is also reported to be essential or non-essential in context of Mtb growth and survival. ", "section_name": "Drug Target Identification", "section_num": null }, { "section_content": "The IPW data has been posted on http://sysborg2.osdd.net, the semantic web-based platform of Open Source Drug Discovery (OSDD) project [47].For ease of access and search, the data is provided through a web-based server available at http://crdd.osdd.net/servers/ipwbuilt using PHP and Mysql.This also works as the annotation and curation interface for the community.Any new submission to this web servers requires http://sysborg2.osdd.net open ID for authentication so that appropriate credits may be given to the members submitting updated information. ", "section_name": "Web Server for Accessing and Searching IPW", "section_num": null } ]	[ { "section_content": "We would like to thank Dr. Vipin Singh, Assistant Professor, Amity University, Noida, for very constructive and detailed comments on the manuscript.We also thank Dr. TS Balganesh, CSIR, and Dr. Vani Brahmachari, ACBR, for fruitful discussions on the manuscript.We acknowledge India 800 foundation for support towards rewarding the best contributors with net books, Hewlett-Packard and Sun Microsystems for providing financial and logistics support for the on-site phase of C2D.We would also like to thank Mahanagar Telephone Nigam Limited, Delhi, India, for providing connectivity and the National Knowledge Network (NKN) for providing high-bandwidth support for C2D on-site phase activities.We also acknowledge School of Information Technology, Jawaharlal Nehru University, Delhi, India, for hosting the on-site phase and Dr. Andrew Michael Lynn and his group, School of Information Technology, Jawaharlal Nehru University, and Dr. S Ramachandran, CSIR-Institute of Genomics and Integrative Biology and his group, Delhi, for providing logistics support.We also thank Dr. GPS Raghava, CSIR-Institute of Microbial Technology, Chandigarh, for hosting the IPW web server.The authors thank all the OSDD members for their active participation. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "This work was supported by the Council of Scientific and Industrial Research, India, Funding (Grant No. HCP0001).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "Table S1 The annotations in the data structure format described in Table 1.This data may be searched in customized manner using the IPW web-interface (http://crdd.osdd.net/servers/ipw).(XLSX) ", "section_name": "Supporting Information", "section_num": null } ]
10.1038/s41375-019-0602-x	Long-term efficacy and safety of first-line ibrutinib treatment for patients with CLL/SLL: 5 years of follow-up from the phase 3 RESONATE-2 study	<jats:title>Abstract</jats:title> <jats:p>RESONATE-2 is a phase 3 study of first-line ibrutinib versus chlorambucil in chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Patients aged ≥65 years (<jats:italic>n</jats:italic> = 269) were randomized 1:1 to once-daily ibrutinib 420 mg continuously or chlorambucil 0.5–0.8 mg/kg for ≤12 cycles. With a median (range) follow-up of 60 months (0.1–66), progression-free survival (PFS) and overall survival (OS) benefits for ibrutinib versus chlorambucil were sustained (PFS estimates at 5 years: 70% vs 12%; HR [95% CI]: 0.146 [0.098–0.218]; OS estimates at 5 years: 83% vs 68%; HR [95% CI]: 0.450 [0.266–0.761]). Ibrutinib benefit was also consistent in patients with high prognostic risk (<jats:italic>TP53</jats:italic> mutation, 11q deletion, and/or unmutated IGHV<jats:italic>)</jats:italic> (PFS: HR [95% CI]: 0.083 [0.047–0.145]; OS: HR [95% CI]: 0.366 [0.181–0.736]). Investigator-assessed overall response rate was 92% with ibrutinib (complete response, 30%; 11% at primary analysis). Common grade ≥3 adverse events (AEs) included neutropenia (13%), pneumonia (12%), hypertension (8%), anemia (7%), and hyponatremia (6%); occurrence of most events as well as discontinuations due to AEs decreased over time. Fifty-eight percent of patients continue to receive ibrutinib. Single-agent ibrutinib demonstrated sustained PFS and OS benefit versus chlorambucil and increased depth of response over time.</jats:p>	[ { "section_content": "Chronic lymphocytic leukemia (CLL) predominantly affects older individuals who frequently have comorbidities that may preclude the use of intensive chemoimmunotherapy regimens, such as fludarabine, cyclophosphamide, and rituximab (FCR) [1][2][3].Before establishment of the chlorambucil-based CD20 combinations, single-agent chlorambucil was considered a standard of care in older patients with CLL [4,5]. Ibrutinib is a first-in-class, oral, once-daily inhibitor of Bruton's tyrosine kinase (BTK), which as a single agent has led to prolonged progression-free survival (PFS) and overall survival (OS) in patients with previously treated CLL [6,7].RESONATE-2 is an international phase 3 study evaluating the efficacy and safety of first-line ibrutinib compared with chlorambucil in older patients with CLL or small lymphocytic lymphoma (SLL) [8].The primary analysis (median follow-up of 18.4 months) demonstrated an 84% reduction in the risk of disease progression (PD) or death (as assessed by an independent review committee) and significant improvement in OS for ibrutinib compared with chlorambucil, supporting the initial approval of first-line ibrutinib for CLL/SLL in the United States and for CLL in the European Union, with ibrutinib now approved for CLL in over 90 countries [8][9][10].Data previously reported from this study after a median follow-up of 28.5 months demonstrated a sustained PFS benefit for ibrutinib and improved depth of response over time, with no new unexpected safety concerns [11].Continued long-term study follow-up is important to provide quantitative assessments of response durability, dimensions of patients' well-being, and safety with continuous single-agent ibrutinib treatment to inform clinical practice. Herein, we present the efficacy and safety outcomes for first-line ibrutinib treatment after a median follow-up of 5 years from the RESONATE-2 study.This represents the longest follow-up to date from a phase 3 trial of BTKdirected therapy in the first-line setting for CLL. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Subjects and methods", "section_num": null }, { "section_content": "RESONATE-2 collectively includes the phase 3, openlabel, international, randomized study PCYC-1115 and extension study (PCYC-1116) comparing the efficacy and safety of ibrutinib versus chlorambucil in first-line CLL/ SLL.Detailed methods have been previously reported [8].Briefly, previously untreated patients without chromosome 17p deletion [del(17p)] aged ≥65 years with CLL/SLL requiring therapy per published criteria [12] were randomized in a 1:1 ratio to oral ibrutinib (420 mg once daily) until PD or unacceptable toxicity, or 12 cycles of chlorambucil (0.5 mg/kg, increased up to 0.8 mg/kg as tolerated, on days 1 and 15 of each 28-day cycle).Following confirmation of PD, patients randomized to chlorambucil were eligible to cross over to second-line treatment with ibrutinib. This study was conducted according to principles of the Declaration of Helsinki and the International Conference on Harmonisation Guidelines for Good Clinical Practice and was approved by the institutional review boards of participating institutions.All patients provided written informed consent.This study was registered with ClinicalTrials.gov,numbers NCT01722487 and NCT01724346. ", "section_name": "Study design and population", "section_num": null }, { "section_content": "Endpoints included PFS, OS, overall response rate (ORR), improvement in hematologic parameters, patient-reported outcomes, and safety.Long-term PD and response were assessed by the investigator per 2008 International Workshop on CLL (iwCLL) criteria [12].Long-term safety data are reported for patients who were initially randomized to ibrutinib.Nonhematologic adverse events (AEs) were graded using Common Terminology Criteria for Adverse Events, v4.03 [13].Hematologic AEs were graded using iwCLL criteria [12]. ", "section_name": "Endpoints and assessments", "section_num": null }, { "section_content": "PFS and OS were analyzed according to the Kaplan-Meier method.To adjust for the impact of crossover on OS, sensitivity analyses were performed as previously described [14]. ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "Requests for access to individual participant data from clinical studies conducted by Pharmacyclics LLC, an AbbVie Company, can be submitted through Yale Open Data Access Project site at http://yoda.yale.edu. ", "section_name": "Data sharing statement", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "A total of 269 patients were randomized to ibrutinib (n = 136) or chlorambucil (n = 133; Supplementary Fig. 1).As previously reported, baseline characteristics were well balanced across treatment arms (Table 1) [8].Treatment with first-line ibrutinib was ongoing in 79 (58%) patients after a median follow-up of 60 months (range, 0.1-66 months), while 56 (41%) discontinued treatment (Table 2).Of 133 patients randomized to chlorambucil, 96 experienced PD (75 crossed over to ibrutinib and 21 did not cross over after PD), one crossed over to receive ibrutinib without documented PD, and 36 patients remained on the chlorambucil arm without PD.Of 21 patients who did not cross over after PD (cross over to ibrutinib was not mandatory), six died, six were still on study without crossing over, and six discontinued the study.Of the 36 patients remaining on the chlorambucil arm without PD, 16 were still on study, 11 discontinued the study, and nine died. ", "section_name": "Patients", "section_num": null }, { "section_content": "Ibrutinib significantly prolonged PFS compared with chlorambucil (median not reached vs 15.0 months [95% confidence interval (CI): 10.2-19.4]),with an 85% reduction in the risk of PD or death (hazard ratio, 0.146 [95% CI: 0.098-0.218];Fig. 1).At 5 years, 70% of patients treated with ibrutinib and 12% with chlorambucil were estimated to be progression-free and alive.Eight (6%) of 136 patients discontinued ibrutinib due to PD. A PFS benefit for ibrutinib over chlorambucil was observed across all patient subgroups examined, including those with the high-risk prognostic features of TP53 mutation, chromosome 11q deletion (del[11q]), and/or unmutated immunoglobulin heavy chain variable region (IGHV) (PFS: HR 0.083 [95% CI: 0.047-0.145])(Fig. 2).When examined individually, the presence of del(11q) and unmutated IGHV were each also associated with increased PFS in ibrutinib-treated patients compared with chlorambucil-treated patients.Ibrutinib dramatically reduced the risk of PD or death by 97% compared with chlorambucil in patients with del(11q) (Fig. 3a).Ibrutinib reduced the risk of PD or death by 90% and 85% compared with chlorambucil for patients with either unmutated and mutated IGHV, respectively (Fig. 3b).PFS was not significantly different for ibrutinib-treated patients with unmutated and mutated IGHV.In patients treated with ibrutinib, 79% of patients with del(11q) and 67% of patients with unmutated IGHV were estimated alive and progression-free at 5 years.Though patients with del(17p) CLL were excluded from the study, 12 ibrutinib-treated patients had TP53 mutation; median PFS was not reached for ibrutinib-treated patients with TP53 mutation or TP53 wild type (HR [95% CI: 0.866 [0.264-2.846])and the 5-year estimates were 56% and 73%, respectively.Only three patients randomized to chlorambucil had TP53 mutation so no comparison could be made between treatments. Median OS was not reached for either the ibrutinib or chlorambucil arms (HR [95% CI], 0.450 [0.266-0.761]).OS estimates at 5 years were 83% for ibrutinib and 68% for chlorambucil without censoring for crossover from chlorambucil to ibrutinib and were 80% for chlorambucil after censoring for crossover to ibrutinib treatment after PD (Supplementary Table 1).In patients with high prognostic risk CLL (TP53 mutation, del[11q], and/or unmutated IGHV), OS at 5 years without censoring for crossover to ibrutinib was 84% for ibrutinib and 62% for chlorambucil (HR 0.376 [95% CI: 0.180-0.786]). ", "section_name": "Progression-free survival and overall survival", "section_num": null }, { "section_content": "With a median follow-up of 5 years (up to 66 months), the ORR including partial response with lymphocytosis was 92% for patients treated with ibrutinib compared with 37% for patients treated with chlorambucil.The proportion of patients with a best response of CR or CR with incomplete marrow recovery (CRi) increased over time (Fig. 4).In the ibrutinib arm, investigator-assessed CR/CRi rates increased from 11% at the primary analysis (median follow-up, 18 months) to 30% after a median follow-up of 5 years.In the ibrutinib arm, ORR and CR rates for patients with highrisk features (i.e., del[11q] or unmutated IGHV) were consistent with the rates seen for all patients treated with ibrutinib. ", "section_name": "Overall response", "section_num": null }, { "section_content": "Among patients with baseline cytopenias treated with ibrutinib, the proportion of patients with sustained hematologic improvement increased over time.Significantly more ibrutinib-treated patients with baseline anemia (hemoglobin ≤11 g/dL) had sustained improvement in hemoglobin levels compared with chlorambucil (90% [46 of 51] vs 45% [25 of 55]; P < 0.0001).Similarly, significantly more patients with baseline thrombocytopenia (platelets ≤100 × 10 9 /L) had sustained improvement in platelet counts after ibrutinib treatment compared with chlorambucil (89% [31 of 35] vs 46% [13 of 28]; P = 0.0007). For patients treated with ibrutinib, median hemoglobin level was 11.6 g/dL at treatment initiation, 13.2 g/dL at year 1, and 13.7 g/dL at year 5 (Fig. 5).Median platelet counts for patients treated with ibrutinib were 143 × 10 9 /L at treatment initiation, 155 × 10 9 /L at year 1, and 147 × 10 9 /L at year 5 (Fig. 5). ", "section_name": "Sustained hematologic improvement", "section_num": null }, { "section_content": "Patient-reported outcomes as assessed with the EQ-5D-5L and FACIT-F were improved with ibrutinib versus chlorambucil.Clinically meaningful improvement in the EQ-5D-5L UIS (≥0.084) [15] and in EQ-5D-5L VAS were observed significantly more frequently with ibrutinib than with chlorambucil (60% vs 44% of patients, P = 0.0089; 65% vs 52%, P = 0.0329, respectively), though no significant difference between ibrutinib-and chlorambucil-treated patients was observed for the proportion of patients who had clinically meaningful improvement in FACIT-F (63% vs 53%).By repeated measure analysis, ibrutinib resulted in significantly greater score improvements over time in EQ-5D-5L UIS (P = 0.0079), EQ-5D-5L VAS (P = 0.0003), and FACIT-F (P = 0.0018) (Supplementary Fig. 2).There were no differences in EQ-5D-5L UIS, EQ-5D-5L VAS, or FACIT-F after chlorambucil-treated patients crossed over to ibrutinib following PD compared with before chlorambuciltreated patients crossed over (Supplementary Fig. 3).Consistent with patient-reported outcomes, when compared with chlorambucil, ibrutinib resulted in higher rates of improvements in disease-related symptoms of weight loss, fatigue, fever, night sweats, anorexia, and abdominal discomfort due to splenomegaly (Supplementary Fig. 4). ", "section_name": "Patient-reported outcomes and disease-related symptoms", "section_num": null }, { "section_content": "At data cutoff, the median duration of ibrutinib treatment was 57 months (range, 0.7-66 months) (Table 2) and median relative dose intensity was 98%.The most frequent AEs of any grade with ibrutinib were diarrhea (50%), cough (36%), and fatigue (36%) (Table 3), and the prevalence of many AEs decreased with time on treatment. Among AEs of particular interest, including those identified during early ibrutinib clinical development, hypertension of any grade occurred in 35 (26%) patients, including 13%, 6%, 6%, 5%, and 7% of patients in years 0-1, 1-2, 2-3, 3-4, and 4-5, respectively (Supplementary Fig. 5).Grade 3 hypertension occurred in 12 (9%) patients, with no grade 4 or 5 events.Atrial fibrillation of any grade occurred at any time in 22 (16%) patients, including 6%, 1%, 6%, 3%, and 4% of patients in years 0-1, 1-2, 2-3, 3-4, and 4-5, respectively (Supplementary Fig. 5).Grade 3 atrial fibrillation occurred in 7 (5%) patients, with no grade Fig. 4 Overall response rate per investigator assessment with first-line ibrutinib.Cumulative best response over time in all patients.Percentages of patients in each category of response may not total the overall proportion with a response because of rounding.CR complete response, CRi complete response with incomplete marrow recovery, nPR nodular partial response, ORR overall response rate, PR partial response, PR-L partial response with lymphocytosis, SD stable disease 4 or 5 events.Major hemorrhage (grouped terms) events occurred in 15 (11%) patients, including 4%, 3%, 1%, 3%, and 2% of patients in years 0-1, 1-2, 2-3, 3-4, and 4-5, respectively (Supplementary Fig. 5).Grade 3 major hemorrhage occurred in 8 (6%) patients, grade 4 in 1 (1%) patient, and no grade 5 events occurred.Of the patients who experienced grade ≥3 major hemorrhage, 6 (67%) were taking concomitant anticoagulation therapy. Treatment discontinuations decreased over time on ibrutinib, with 7% of patients discontinuing because of AEs in years 0-1, 6% in years 1-2, 5% in years 2-3, 6% in years 3-4, and 1% in years 4-5 (Supplementary Fig. 6).Thirtyeight patients experienced AEs leading to discontinuation of ibrutinib; those reported in ≥2 patients were atrial fibrillation (n = 4), and palpitations, pneumonia, and worsening CLL (n = 2 each), in addition to 2 deaths with unknown cause. The rates of dose reductions due to AEs were similar over time (Supplementary Fig. 6), with rates of 9% for patients in years 0-1, 8% in years 1-2, 6% in years 2-3, 5% in years 3-4, and 7% in years 4-5.Dose reductions due to any-grade AEs occurred in 27 patients; 25 (93%) had improvement or resolution of the AE following dose reduction.At the time of data cutoff, 12/27 remained on ibrutinib and 15/27 had discontinued ibrutinib at any time during follow-up after the dose reduction.Reasons for subsequent discontinuation at any time during follow-up were for any AE (n = 9; two patients had dose reduction and then discontinued because of the same AE), withdrawal from the study (n = 3), PD (n = 2), and physician decision (n = 1).AEs leading to dose reduction reported in >1 patient were thrombocytopenia (n = 3), and anemia, arthralgia, diarrhea, fatigue, and palpitations (n = 2 each).After AE-related dose reductions, ibrutinib dose was successfully re-escalated back to previous dose for ≥2 treatment cycles in six (22%) patients, and re-escalated treatment lasted a median of 589 days (preceding dose reductions lasting a median of 103 days). Ibrutinib dosing was held for ≥7 consecutive days because of any grade AEs in 70 patients and 60 patients had complete resolution of the AE following dose hold.At the time of data cutoff, 36/70 of these patients remained on ibrutinib and 34/70 had discontinued ibrutinib at any time during follow-up after the dose hold.Reasons for subsequent discontinuation at any time during follow-up included any further AE (n = 20), withdrawal from study (n = 6), PD (n = 5), death (n = 2), or physician decision (n = 1).Of note, only six patients discontinued due to the same AE after the dose hold (atrial fibrillation, cerebral hemorrhage, cognitive disorder, myelodysplastic syndrome, non-small cell lung cancer, and staphylococcal sepsis).For these 70 patients, the median duration between first dose hold of ibrutinib to study treatment discontinuation or last known date alive for those still on treatment was 48 months (maximum 64+ months).Following dose hold, ibrutinib was restarted at the same dose in 42 patients and at a reduced dose in 22 patients. At the time of data cutoff, 23 patients randomized to ibrutinib died (8 while on treatment); 4 due to PD (all were aged ≥70 years).One AE of pneumonia was considered possibly related to ibrutinib.There were six patients for Fig. 5 Hemoglobin levels and platelet counts over time in ibrutinib-treated patients.The horizontal line represents the lower limit of normal for platelet counts.CI confidence interval whom cause of death was unknown, and of the remaining, there were two infections, three second malignancies, and one each reported as multiorgan failure, heart attack, sudden death, heart failure, sepsis, pulmonary fibrosis, and septic shock (Supplementary Fig. 7). ", "section_name": "Safety", "section_num": null }, { "section_content": "Outcomes following discontinuation of first-line ibrutinib treatment are shown in Supplementary Table 2. Median OS following discontinuation was not reached (range, 0-64+ months) in patients who discontinued ibrutinib because of AEs (n = 29).Only eight patients discontinued ibrutinib because of PD (including two patients due to Richter's transformation); of these patients, 50% are still alive or had exited study with no known death at the data cut.The median OS following ibrutinib discontinuation due to PD was 20 months (range, 1+ to 28 months).Median PFS for patients who were in CR/CRi at ibrutinib discontinuation was 56 months (95% CI: 44, NE) compared with 33 months (95% CI: 26, 46) for patients who were not CR/CRi at ibrutinib discontinuation (HR [95% CI]: 0.390 [0.118, 1.285]). Of patients with available follow-up data after ibrutinib discontinuation, 14 patients received subsequent therapy for CLL, including standard chemoimmunotherapy (FCR, BR, or GC) (n = 8), chemotherapy (n = 3), and novel agents (n = 3).Of nine patients with best overall response to subsequent therapy reported, seven responded, one had stable disease, and one had PD.Eleven of the 14 patients remained alive at last follow-up, two patients withdrew consent, and one patient died. ", "section_name": "Outcomes following ibrutinib discontinuation", "section_num": null }, { "section_content": "With long-term follow-up of the RESONATE-2 study, single-agent ibrutinib continues to demonstrate significant and durable clinical benefit in older patients, including those with high-risk prognostic features (TP53 mutation, del(11q), and/or unmutated IGHV).No new safety signals emerged over the extended treatment duration, and many events decreased over time.This is the longest follow-up report of patients receiving first-line treatment with a BTK inhibitor in a phase 3 study to date. With up to 66 months of follow-up, the median investigator-assessed PFS per iwCLL criteria was not reached in the ibrutinib arm and was 15 months (95% CI [10][11][12][13][14][15][16][17][18][19]) in the chlorambucil arm.The rate of PD during ibrutinib treatment was low; only 8/136 (6%) patients progressed while receiving ibrutinib, implying a low rate of developing the BCR pathway mutations associated with PD.Improvement in PFS with first-line ibrutinib compared with chlorambucil remains durable, as evidenced by an 85% reduction in the risk of progression or death.Accordingly, when comparing similar timepoints between studies, the 3year PFS rate with ibrutinib (82%) was higher than that with chlorambucil (25%) and appears higher than rates previously reported for first-line chemoimmunotherapy with FCR (70%) or BR (55%) in an older patient population than in the CLL10 study, although the proportion of patients with unmutated IGHV was higher in CLL10 than in the current study [16,17].Three-year PFS rates for ibrutinib also appeared favorable compared with chlorambucil in combination with obinutuzumab (<40%) in older, less fit patients in the CLL11 study [5]. In addition, ibrutinib substantially improved PFS in highrisk patients with del(11q) or unmutated IGHV compared with chlorambucil.Whereas the presence of del(11q) [18] or unmutated IGHV [16,19] confers poor outcomes in the chemoimmunotherapy setting, we observed prolonged PFS with ibrutinib compared with chlorambucil in patients with del(11q) or with unmutated IGHV.Patients with the composite high prognostic risk genomics of del(11q), unmutated IGHV, or TP53 mutation experienced markedly improved PFS with ibrutinib, with a 92% reduction in risk of PD or death versus chlorambucil.However, there were no meaningful differences in PFS for ibrutinib-treated patients when these high-risk factors were evaluated individually (del[11q], unmutated IGHV, or TP53 mutation).The exclusion of patients with del(17p) is an important limitation of this analysis given its prognostic significance and frequent overlap with TP53 mutations [20].Taken together, our results suggest that these high-risk prognostic features may not have meaningful prognostic value with ibrutinibmediated inhibition of the BCR pathway, unlike with chemoimmunotherapy. Cross-study analyses suggest that the clinical benefit is highest when ibrutinib is used as initial therapy versus as a later line of therapy [7], and the present study demonstrates the durability of those outcomes in the first-line setting with more than 5 years follow-up.Real-world studies of patients receiving first-line treatment, including patients who would have been excluded from RESONATE-2 due to age <65 years or the presence of del(17p), demonstrate similar response rates (71-82% vs 92%) and proportion of discontinuations due to AEs (51-63% vs 52% of patients who discontinued) as the present study [21][22][23][24]. Additional randomized trials have found that adding rituximab to ibrutinib does not increase PFS compared with single-agent ibrutinib in the first-line setting [25] and in relapsed/refractory patients or high-risk patients with del(17p) or TP53 mutations receiving first-line treatment [26].In the iLLUMINATE study of ibrutinib plus obinutuzumab, median PFS was also not reached for patients with del(11q) nor for patients with unmutated IGHV, similar to what we report here for single-agent ibrutinib (Fig. 3).These results with del(11q) and IGHV mutational status are also consistent across two other phase 3 studies of ibrutinib in different patient populations and as a single agent or in combination regimens [6,18,27].A pooled analysis of three phase 3 randomized studies (RESONATE, RESO-NATE-2, and HELIOS) further showed that the prognostic risk factors of del(11q) and unmutated IGHV traditionally associated with worse outcomes in patients with CLL have less prognostic significance with ibrutinib therapy in patients without del(17p) [28]. Our results also demonstrate an improved depth of response over time with first-line ibrutinib.Investigatorassessed CR/CRi rates in ibrutinib-treated patients improved from 11% at the primary analysis (median followup 18.4 months) [8] to 30% after a median of 5 years follow-up.In addition, we continue to observe sustained improvement in anemia and thrombocytopenia with ibrutinib, which are important and frequent reasons patients with CLL initiate treatment.These improvements may help alleviate the fatigue that is a major component of symptom burden and reduced quality of life in patients with CLL [29], especially those with advanced age and multiple comorbidities [29].Given that patients with CLL may remain on ibrutinib for many years, durable improvement in quality of life during treatment is an important goal.In this study, ibrutinib treatment improved patient-reported outcomes and disease-related symptoms that were sustained through extended follow-up, in contrast to the worsened quality of life outcomes reported by patients treated with chlorambucil, as PD occurred earlier and more frequently. As the majority of patients with CLL (including those in this study) are elderly and may be less tolerant of toxicities, treatments with a tolerable safety profile in long-term use are essential.Late high-grade toxicities have been observed with other CLL therapies [30].In this study, no unexpected AEs were identified after extended follow-up of ibrutinibtreated patients.Patients continued to experience new AEs throughout extended treatment, with some AEs appearing as late events, such as cataracts, fall, and herpes zoster, although the impact of aging in this patient population with extended follow-up cannot be fully accounted for.In the primary analysis of RESONATE-2 (median follow-up, 18 months), the three most common AEs were diarrhea (42%), fatigue (30%), and cough (22%) [8], while in the current analysis at median of 5 years of follow-up (~3.3fold longer exposure to ibrutinib) these AEs continue to be the most common events in 50%, 36%, and 36% of patients, respectively.Compared with a younger enrolled patient population treated with ibrutinib plus rituximab for a shorter median follow-up of 33 months in the phase 3 ECOG1912 study, more ibrutinib-treated patients in RESONATE-2 (~1.8-fold longer exposure to ibrutinib) experienced grade ≥3 AEs (83% vs 58%) overall, although there was a lower frequency of grade ≥3 AEs in the ibrutinib plus rituximab cohort compared with the FCR cohort in ECOG1912 (58% vs 72%) [31].Compared with a similar patient population in the ALLIANCE study, fewer ibrutinib-treated patients in RESONATE-2 experienced grade ≥3 hypertension than ibrutinib-treated patients in the ALLIANCE study (8% vs 29%), despite the difference in follow-up (median: 60 months vs 38 months, respectively) [25]. Overall, many AEs decreased over time in this study, with some exceptions, such as hypertension (the prevalence of grade ≥3 hypertension remained stable over time).The incidence of major hemorrhage was generally highest in the first 2 years of treatment and decreased thereafter.Based on prior reports, the risk for bleeding with ibrutinib is most often observed within the first 12 months of treatment and then decreases over time [27,32,33].Similarly, atrial fibrillation typically occurs early after ibrutinib initiation and remains constant or declines over time [7,[34][35][36].In this study after a median follow-up of 5 years, the cumulative rate of major hemorrhage increased from 4% at the primary analysis to 11%, and for atrial fibrillation increased from 6% to 16%; however, few patients required dose reduction or discontinued because of these AEs.Overall, dose reductions and discontinuations due to AEs were infrequent and discontinuations due to AEs decreased over time with continued treatment, with 73% of patients receiving ibrutinib for >3 years.The ongoing incidence of new AEs and increasing prevalence of hypertension highlights the importance of ongoing follow-up and monitoring during treatment to maximize optimal management of AEs with dose modifications (dose holds and reductions) and thus mitigate the impact of AEs and enable patients to continue to benefit from ongoing first-line ibrutinib. As novel agents continue to be developed for CLL, longterm data are crucial to inform practice.Additional BTK inhibitors in development for CLL have shown encouraging efficacy, but results of randomized comparative studies are not yet available and these agents lack long-term safety and efficacy data [37,38].Here, we demonstrated that with a median of 5 years of follow-up, over half of patients with CLL/SLL were able to receive long-term continuous firstline treatment with single-agent ibrutinib and had sustained efficacy benefits (70% of ibrutinib-treated patients estimated progression-free), including-importantly-in patients with high-risk prognostic features, such as del(11q) or unmutated IGHV.and travel expenses for Janssen and Gilead.HM reported honoraria and travel expenses from AbbVie, Janssen, Novartis, and Roche; and a consultancy/advisory role with Janssen.DS reported honoraria from Celgene, Roche, MSD, and Janssen; travel expenses from Celgene, Bristol-Myers Squibb, Janssen, and Novartis; and research funding from Amgen, MSD, Pharmacyclics LLC, an AbbVie Company, AbbVie, Sanofi, Roche, BeiGene, and Acerta.CM reported a consulting/advisory role with Pharmacyclics LLC, an AbbVie Company, Janssen, and AbbVie.SD reported employment with Pharmacyclics LLC, an AbbVie Company; and stock ownership with AbbVie, Celgene, Gilead, GSK, and Exelixis.IL reported employment with Pharmacyclics, an AbbVie Company, and spouse employment with The Permanente Medical Group; and stock ownership with AbbVie, Gilead Sciences, Clovis, Infinity, The Permanente Medical Group, and Reviva Pharmaceuticals.JPD reported employment with Pharmacyclics LLC, an AbbVie Company, and CTI BioPharma Corp; and stock ownership with AbbVie and CTI BioPharma Corp. TJK reported a consultancy/advisory role for AbbVie, Genentech-Roche, Gilead, Pharmacyclics LLC, an AbbVie Company, and Celgene; and research funding from AbbVie, Genentech-Roche, Pharmacyclics LLC, an AbbVie Company, and Oncternal.SG and FO reported no relevant financial disclosures. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. ", "section_name": "Discussion", "section_num": null } ]	[ { "section_content": "Acknowledgments We thank the patients who participated in the study and their supportive families, as well as the investigators and clinical research staff from the study centers.This study was sponsored by Pharmacyclics LLC, an AbbVie Company.Medical writing support was provided by Valerie Hilliard, PhD, and funded by Pharmacyclics LLC, an AbbVie Company.role with Janssen; speakers' bureau and travel expenses from Gilead, Janssen, Novartis, Pharmacyclics LLC, an AbbVie Company, and TG Therapeutics; and research funding from BeiGene, Gilead, Pharmacyclics LLC, an AbbVie Company, and TG Therapeutics.PMB reported a consultancy/advisory role for AbbVie and Pharmacyclics LLC, an AbbVie Company; and research funding from Pharmacyclics LLC, an AbbVie Company.TR reported research funding from Pharmacyclics LLC, an AbbVie company.CO reported honoraria from Gilead, Janssen, AbbVie, Roche, Merck, Teva, and AstraZeneca.PG reported honoraria from AbbVie, Acerta, BeiGene, Gilead, Roche, Sunesis, Celgene, and Janssen; a consultancy/advisory role from AbbVie, Acerta, BeiGene, Gilead, Janssen, Pharmacyclics LLC, an AbbVie Company, Celgene, and Sunesis; speakers' bureau for Gilead; and research funding from AbbVie, Gilead, Janssen, and Novartis.AT reported a consultancy/advisory role for Janssen spa, Gilead, Sunesis, and AbbVie; and speakers' bureau for Janssen.OB reported a consultancy/advisory role for AbbVie and research funding from Janssen.PH reported honoraria, a consultancy/advisory role, and research funding from Janssen, Pharmacyclics LLC, an AbbVie Company, and AbbVie; and travel expenses from Janssen and AbbVie.SEC reported honoraria from Pharmacyclics LLC, an AbbVie Company, and Janssen; a consultancy/advisory role for AbbVie, Astellas, AstraZeneca, Celgene, Genentech, Pharmacyclics LLC, an AbbVie Company, Novartis, and Janssen; travel, accommodations, expenses from Abb-Vie, BeiGene, Celgene, Genentech, Janssen, Pharmacyclics LLC, an AbbVie Company; expert testimony for Genentech; other relationship with BeiGene; and research funding from Celgene, Gilead, Janssen, Pharmacyclics LLC, an AbbVie Company, AbbVie, Takeda, and Acerta.SD reported a consultancy/advisory role for AbbVie, Janssen, GlaxoSmithKline, and Bristol-Myers Squibb; and speakers' bureau ", "section_name": "", "section_num": "" }, { "section_content": "Author contributions JAB, JPD, and TK designed the study or current analysis.JAB, PMB, TR, CO, PG, AT, OB, PH, SEC, SD, SG, HM, DS, FO, and CM contributed to collecting the data with the other RESONATE-2 investigators and their research teams.All authors had full access to the data, were involved in data interpretation, and vouch for the accuracy of the data.JAB, SD, IL, and JPD collaboratively wrote the first draft of the manuscript.All authors reviewed and revised the manuscript, provided final approval, and made the decision to submit the manuscript for publication.Medical writing support was provided by a professional medical writer and funded by the sponsor. Conflict of interest JAB reported honoraria and a consulting/advisory ", "section_name": "Compliance with ethical standards", "section_num": null }, { "section_content": "Author contributions JAB, JPD, and TK designed the study or current analysis.JAB, PMB, TR, CO, PG, AT, OB, PH, SEC, SD, SG, HM, DS, FO, and CM contributed to collecting the data with the other RESONATE-2 investigators and their research teams.All authors had full access to the data, were involved in data interpretation, and vouch for the accuracy of the data.JAB, SD, IL, and JPD collaboratively wrote the first draft of the manuscript.All authors reviewed and revised the manuscript, provided final approval, and made the decision to submit the manuscript for publication.Medical writing support was provided by a professional medical writer and funded by the sponsor. ", "section_name": "", "section_num": "" }, { "section_content": "Conflict of interest JAB reported honoraria and a consulting/advisory ", "section_name": "Compliance with ethical standards", "section_num": null } ]
10.1186/s40364-016-0057-4	RECQ helicases are deregulated in hematological malignancies in association with a prognostic value	RECQ helicase family members act as guardians of the genome to assure proper DNA metabolism in response to genotoxic stress. Hematological malignancies are characterized by genomic instability that is possibly related to underlying defects in DNA repair of genomic stability maintenance.We have investigated the expression of RECQ helicases in different hematological malignancies and in their normal counterparts using publicly available gene expression data. Furthermore, we explored whether RECQ helicases expression could be associated with tumor progression and prognosis.Expression of at least one RECQ helicase family member was found significantly deregulated in all hematological malignancies investigated when compared to their normal counterparts. In addition, RECQ helicase expression was associated with a prognostic value in acute myeloid leukemia, chronic lymphocytic leukemia, lymphoma and multiple myeloma.RECQ helicase expression is deregulated in hematological malignancies compared to their normal counterparts in association with a prognostic value. Deregulation of RECQ helicases appears to play a role in tumorigenesis and represent potent therapeutic targets for synthetic lethal approaches in hematological malignancies.	[ { "section_content": "The RECQ family of DNA helicases is a family of conserved enzymes that display highly-specialized and vital roles in the maintenance of genome stability [1]. In humans, RECQ helicase family has five members with similar catalytic core: RECQ1, BLM, WRN, RECQ4 and RECQ5 [1].Mutations in three of the five human RECQ helicases, BLM, WRN and RECQ4, lead to genetic disorders as Bloom, Rothmund-Thompson and Werner's syndromes that are associated with cancer predisposition, premature ageing and developmental abnormalities [1,2].The Bloom's syndrome helicase, BLM has DNA annealing and unwinding activities.Through its interaction with TOPOIIIα, BLM unwinds the short stretches of naked duplex DNA and processes homologous recombination (HR) intermediates containing a double holiday junction [1,2].This helicase appears to prefer specific structures including D-loops and Holliday junctions and promotes Holliday junction branch migration [3].It may suppress hyper-sister chromatid exchange (SCE) by disruption of D-loop recombination intermediates and also might be involved in the suppression of crossing over during homology-mediated recombination [4].BLM-mediated crossover suppression may involve synthesis-dependent strand annealing (SDSA) [3].This helicase facilitates telomere replication by resolving G4 structures [5].Defects in BLM are also associated with the cancer phenotype [4]. Unlike the other members of the RECQ helicase family, the Werner's syndrome helicase, WRN contains both the classical helicase activity and 3'-to 5' exonuclease activity that target multiple DNA or RNA-DNA hybrid structures [1,2].As BLM, WRN appears to have robust in vitro G4 unwinding activity [6] and plays a specialized role in telomere replication by disruption of G-quadruplex stretches [7].Another specific substrate of WRN is D-loop.WRN repress the inappropriate telomeric recombination intermediates through its ability to resolve the D-loops [8].This helicase was found to be involved in the repair of double strand DNA breaks and studies on Werner's syndrome fibroblasts have shown defects in recombination intermediate resolution which suggests that WRN is involved in HR [9].WRN can bind to NBS1 [10], a member of the MRN complex, and to the Ku70/Ku80 heterodimer [11], a core non-homologous end joining DNA repair (NHEJ) complex. RECQ1 is the shortest of the human RECQ family helicases.RECQ1 displays specific functions in branch migration and restart of reversed DNA replication forks upon DNA topoisomerase I inhibition that is not shared by other human RECQ helicases [12,13].Studies have also shown that RECQ1 plays a role in DNA strand breaks repair, mismatch repair and resistance to replication stress [1,2,13,14].RECQ1 can also contribute to tumor development and progression by regulating the expression of key genes that promote cancer cell migration, invasion and metastasis [15]. The gene encoding for RECQ5 helicase was found to be localized on human chromosome 17q23-25, a region associated with both breast and ovarian cancer [16].RECQ5 was found to cause a significant increase in the frequency of spontaneous SCE [1,2].As BLM, RECQ5 was shown to play an essential role in suppression of crossovers [17].RECQ5 was identified as a potential proto-oncogene in mouse leukemia [18].RECQ5 is the only member of RECQ family associated with RNA polymerase II, maintaining genomic stability during transcription [19]. RECQ helicase are at the crossroad between replication, recombination, DNA repair and transcription and could represent potent therapeutic targets for cancer therapy [20].Hematological malignancies are characterized by genomic instability that is possibly related to underlying defects in DNA repair of genomic stability maintenance.Since these helicases play important roles in the maintenance of chromosomal stability [21], we focused on RECQ helicases expression in hematological cancers compared to their normal counterparts and the association with prognostic impact. ", "section_name": "Background", "section_num": null }, { "section_content": "RECQ helicase gene expression levels were analyzed in different types of hematological malignancies and in their normal counterparts using Oncomine Cancer Microarray database [22] as indicated in Table 1.Abnormal expression of at least one RECQ helicase was identified in all analyzed hematological malignancies (Table 1).RECQ1 was found to be significantly overexpressed in mantle cell lymphoma (P = 0.0025) [23], in unspecified peripheral T-cell lymphoma (P = 0.0038) [24], anaplastic large lymphoma (P = 0.0024) [24] and angioimmunoblastic large cell lymphoma (P = 0.001) [24] (Fig. 1a). WRN is significantly overexpressed in primary effusion lymphoma compared to normal B cells (p = 0.003) [23] (Fig. 1a). RECQ4 expression was increased in Burkitt Lymphoma (P = 0.001) [23,25], in diffuse large B cell lymphoma (P = 0.004) [23] and also in primary effusion lymphoma (P = 0.005) [23] compared to normal counterpart (Fig. 1a). RECQ5 is significantly overexpressed in acute myeloid leukemia (P = 0,001) [26] (Fig. 1a). Comparing RECQ helicases expression between normal plasma cells (BMPC), premalignant cells from MGUS patients and multiple myeloma cells (MMC) [27], BLM and WRN were found to be significantly downregulated in MMC compared to normal BMPC (P = 0.002 and P = Table 1 RECQ family member expression in hematological malignancies compared to that of their normal tissue counterparts using publicly available gene expression data, including the Oncomine Cancer Microarray and Genomicscape databases 0.001 respectively) (Fig. 1b).A decreased expression of BLM was also observed in MGUS compared to BMPC (P = 0.01).RECQ1 and RECQ4 are overexpressed in MGUS (P = 0.006 and P = 0.002) and MMC (P < 0.0001 and P = 0.0009) compared to BMPC.Furthermore, a significant increased expression of RECQ1 in MMC compared to MGUS was identified (Fig. 1b). Furthermore, using the human protein atlas database [28][29][30], the expression of RECQ1, RECQ4 and RECQ5 could be confirmed at protein level in myeloid and lymphoid cancer cell lines (Additional file 1: Figure S1). We investigated whether RECQ helicases expression could be associated with tumor progression and prognosis in hematological malignancies (Table 2). In AML patients with abnormal karyotype (Verhaak cohort, N = 521 patients) [31], a high expression of BLM and RECQ4 is associated with a better overall survival (OS) (P = 0.01 and P = 0.003).At the opposite, high RECQ5 expression was linked with a poor prognosis in the same cohort of patient (P = 0.008) (Fig. 2a).In cox multivariate analysis, only RECQ5 expression kept prognostic value (P = 0.01, hazard ratio (HR) = 1.43). In AML with normal karyotype, (Metzeler cohort, N = 78) [32], gene expression of four RECQ helicases out of five were identified to predict for OS.High expression of RECQ1 (P = 0.02), BLM (P = 0.01) and RECQ5 (P = 0.03) were found to be associated with poor prognosis.In contrast, high RECQ4 expression was linked with a better outcome (P = 0.03) (Fig. 2b).When tested together in a cox multivariate analysis, only RECQ4 expression remained significant (P = 0.009; HR = 0.4). Interestingly, RECQ5 overexpression was only identified in myeloid malignancies in association with an adverse prognosis.RECQ5 increased expression was recently reported in JAK2V617F myeloproliferative neoplasms [33].RECQ5 depletion in JAK2V617Fmutant cells impairs replication after hydroxyurea treatment leading to a significant increased doublestranded breaks and apoptosis [33].RECQ5 represents a potent regulator of genome stability in myeloproliferative neoplasms in association with drug resistance a b Fig. 1 Increased RECQ helicase gene expression in hematological malignancies compared to normal counterparts using Oncomine database (a) and Genomicscape database (b).Data sets in a given panel were from the same study.GEP data are log transformed (Oncomine) or not (Genomicscape) and normalized as previously described [64] [33].RECQ5 overexpression could also be involved in AML pathophysiology and chemoresistance. Even if RECQ1 mutations have been recently shown to been associated with predisposition to breast cancer [34,35], no link between RECQ1, BLM, WRN, and RECQ4 deregulation and lymphoid malignancies were previously reported. In chronic lymphocytic leukemia (CLL), a poor prognosis was linked with high RECQ5 expression (P = 8E-8) and a better outcome was associated with high WRN expression (P = 0.0006) (Fig. 3a). In a cohort of patients with follicular lymphoma (FL) (Staudt cohort, N = 180) [36], high RECQ1 and RECQ5 expression represented adverse prognostic factors (P = 0.003 and P = 0.0006 respectively) whereas RECQ4 expression was found to be associated with a good prognosis (P = 0.009) (Fig. 3b).Interestingly, RECQ1, RECQ4 and RECQ5 expresison remained independent when tested in cox multivariate analysis (P < 0.0001; HR = 2.8; P = 0.002; HR = 0.49 and P < 0.01; HR = 1.78 respectively). In diffuse large B cell lymphoma (DLBCL), only RECQ5 expression was associated with a prognostic value.Low RECQ5 expression was a poor prognostic marker in two independent cohorts of patients (P = 0.02 in a cohort of patients treated by combination of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) therapy (N = 181) and P = 0.01 in a cohort of patients treated by Rituximab combined with CHOP (R-CHOP) regimen (N = 233)) (Fig. 3c) [37]. In MM, high RECQ1, WRN and RECQ4 expression are associated with an adverse prognosis in the UAMS cohort treated with total therapy 2 (Fig. 4) [27]. These data demonstrate a link between RECQ helicase expression and a prognostic value in different hematological malignancies. Hematological malignancies are characterized by genomic instability that could be related to defects in DNA repair [21].The RECQ family of DNA helicases is a family of conserved enzymes that display highly specialized and vital roles in the maintenance of genome stability.Mutations in three of the five human RECQ helicases, BLM, WRN and RECQL4, are associated with genetic disorders characterized by chromosomal instability and increased susceptibility to cancer including leukemia [38,39].Mutations in BLM result in a dramatic lowering of BLM mRNA levels and premature termination of protein translation owing to nonsense-mediated mRNA decay [1,40].Patients with Bloom syndrome exhibit cancer predisposition including most types of cancers and particularly non-Hodgkin's lymphoma, leukemias and carcinomas of skin, breast and colon [1].Interestingly, a low BLM expression is associated with a poor prognosis in AML with complex karyotypes (Fig. 2a).The cancer spectrum observed in patients with Werner's syndrome is characterized mainly by cancers of mesenchymal origin and some epithelial cancers [1,41].RECQ4 mutations are found in Rothmund-Thomson syndrome, RAPADILINO syndrome and Baller-Gerold syndrome [1,41].Rothmund-Thomson syndrome are characterized by predisposition to mainly osteosarcoma whereas RAPADILINO syndrome are linked with lymphoma and osteosarcoma predisposition [1]. Specific recurrent chromosomal translocations have been associated with DNA repair deficiencies linked with repression of DDR (DNA damage response) genes in AML [42].In PML-RARA, PML and BLM are delocalized from the nuclear bodies into microspeckled nuclear regions [43].All trans retinoic acid (ATRA) treatment of APL patients leads to degradation of PML-RARA and relocalization of BLM to nuclear bodies [43] suggesting that PML-RARA are involved in genomic instability in APL through disruption of BLM and PML localization and activity.Interestingly, we reported that low BLM and RECQ4 expression are associated with a poor prognosis in AML with abnormal karyotype (Fig. 2a), suggesting that downregulation of RECQ helicases could be involved in leukemogenesis and genomic instability.At the opposite, in AML with normal karyotype, RECQ1, BLM and RECQ5 high expression are associated with a poor prognosis (Fig. 2b).As reported in solid cancer, RECQ helicase overexpression could be a marker of chemoresistance and higher GOOD : A high RECQ helicase expression is associated with a better outcome BAD : A high RECQ helicase expression is associated with a poor prognosis proliferation helping AML cells to deal with replication stress [44,45].B lymphocytes are continuously produced during adult life and they undergo different genetic alterations associated with DNA breaks, including VDJ recombination, Ig class switch recombination and somatic hypermutation.These mechanisms must be tightly regulated to prevent tumorigenesis and ensure efficient immune response [46].Collapsed DNA replication forks occurring in rapidly dividing lymphocytes leads to a restart failure and results in an interruption of the normal developmental program [47].HR is required for lymphoid development [47].Aberrations a b affecting HR actors are correlated with genomic instability in B cell cancers [48].By their involvement in HR and also by their ability to resolve and to continue the normal fork replication after DNA damage or replication fork arrest, WRN [49], BLM [4], RECQ1 [12] and RECQ5 [50] helicases might be crucial in lymphoid development and aberration in their expression or function can lead to cancer genesis.Interestingly, low expression of WRN in CLL and FL, low RECQ4 expression in FL and low RECQ5 in DLBCL are associated with a poor prognosis (Fig. 3).Furthermore, high RECQ5 expression in CLL and FL and high RECQ1 expression in FL are associated with a poor prognosis and could be involved in chemoresistance.In lymphoma, deregulation of DDR is associated with tumorigenesis [51,52], poor prognosis [53,54] and could represent a potent therapeutic target [53,55,56]. In MM, patients with extensive chromosomal instability and replicative stress are associated with an adverse outcome [27,[57][58][59].Accordingly, high RECQ1, WRN and RECQ4 expression is associated with a significant poor survival in MM patients (Fig. 4).Although WRN was found to be significantly downregulated in MMC compared to normal BMPC (Fig. 1b), patients with high expression display a poor prognosis.WRN is located on chromosome 8p deleted in % of MM patients without prognostic value [60].These data could explain the significant downregulation of WRN expression in MM compared to normal BMPC. Recently, a set of molecule inhibitors of WRN and BLM was characterized [61,62].These new molecules could open up new therapeutic strategies for targeting hematological malignancies characterized by RECQ helicase deregulation and a poor prognosis. ", "section_name": "Results and discussion", "section_num": null }, { "section_content": "The analysis reported here demonstrates that RECQ helicase expression is deregulated in hematological malignancies compared to their normal counterparts in association with a prognostic value in AML, CLL, lymphoma and MM.Deregulation of RECQ helicases appears to play a role in tumorigenesis and could be involved in genomic instability and chemoresistance in hematological malignancies.RECQ helicases represent potent therapeutic targets for synthetic lethal approaches. ", "section_name": "Conclusion", "section_num": null }, { "section_content": "Databases: We used Oncomine Cancer Microarray database (http://www.oncomine.org)[22] and Genomicscape (http://genomicscape.com/) [63] to study gene expression of RECQ family members in nine different human hematological malignancies and their normal tissue counterpart as indicated in Table 1.To compare the gene expression of a tumor type to its normal counterpart, we used gene expression data from a same study with the same methodology.All data were log transformed, median centered per array, and the standard deviation was normalized to one per array [22]. Statistical comparisons were done with Mann-Whitney or Student t-test as previously published [64]. Prognosis values of each member of RECQ family in hematological malignancies were determined by using Maxstat R package based on publicly available data (Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/);accession numbers GSE6891, GSE12417, GSE22762, GSE16131, GSE10846 and GSE4581) analyzed with Genomicscape [63] as previously reported [65]. ", "section_name": "Methods", "section_num": null } ]	[ { "section_content": "This work was supported by grants from French INCA (Institut National du Cancer) Institute (2012-109/087437), Languedoc Roussillon CRLR (R14026FF), Fondation de France (201400047510), ITMO Cancer (MM&TT) and AXLR SATT (30041633).EV is supported by a grant from Guillaume Espoir association (Saint-Genis-Laval, France). ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "12 No No No No YES B-cell neoplasms Primary Effusion Lymphoma 8 No No YES YES No Chronic Lymphocyte Leukemia 11 No No YES No No Diffuse Large B-cell Lymphoma 8 No No No YES No Burkitt's Lymphoma 8 No No No YES No Mantle Cell Lymphoma 11 YES No No No No Multiple Myeloma 1 YES YES YES YES No T-cell neoplasms Unspecified Peripheral T-cell Lymphoma 11 YES No No No No Anaplastic Large Cell Lymphoma 11 YES No No No No Angioimmunoblastic T-Cell Lymphoma 11 YES No No No No ", "section_name": "", "section_num": "" }, { "section_content": "Additional file 1: Figure S1.RECQ helicase gene and protein expression in myeloid and lymphoid cell lines using using the human protein atlas database.RECQ1, RECQ4 and RECQ5 expression could be confirmed at protein level in myeloid and lymphoid cancer cell lines.(PDF 599 kb) The authors declare that they have no competing interests. EV performed research, bioinformatics, and participated in the writing of the paper.AK participated in the bioinformatics.PP participated in the writing of the paper.BK participated in the research and in the writing of the paper.JM supervised the research, bioinformatics, and the writing of the paper.All authors read and approved the final manuscript. ", "section_name": "Additional file", "section_num": null }, { "section_content": "Additional file 1: Figure S1.RECQ helicase gene and protein expression in myeloid and lymphoid cell lines using using the human protein atlas database.RECQ1, RECQ4 and RECQ5 expression could be confirmed at protein level in myeloid and lymphoid cancer cell lines.(PDF 599 kb) ", "section_name": "Additional file", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "EV performed research, bioinformatics, and participated in the writing of the paper.AK participated in the bioinformatics.PP participated in the writing of the paper.BK participated in the research and in the writing of the paper.JM supervised the research, bioinformatics, and the writing of the paper.All authors read and approved the final manuscript. ", "section_name": "Authors' contributions", "section_num": null } ]
10.1038/s41419-018-1043-6	Akt2 mediates glucocorticoid resistance in lymphoid malignancies through FoxO3a/Bim axis and serves as a direct target for resistance reversal	<jats:title>Abstract</jats:title><jats:p>Glucocorticoids (GCs) are widely used drugs in the treatment of lymphoid malignancies; resistance of GCs in lymphocytes confers poor prognosis and the mechanisms are poorly understood. Here, we found T-acute lymphoblastic leukemia (T-ALL) cells acquire resistance to dexamethasone (DEX)-mediated killing through abnormal activation of Akt, resulting in inhibition of the FoxO3a/Bim pathway. The resistant state was reported to be associated with increased glycolysis, NOTCH1 activating mutations and activated PI3K/ serum GS regulated kinases (SGK) pathway. Use of aforementioned pathway inhibitors blocked FoxO3a-phosphorylation and partially improved DEX-mediated killing of GC-resistant T-ALL cells, further revealing the essential role of the FoxO3a/Bim pathway in the development of GC resistance. Inhibition of Akt is most effective at restoring sensitivity to DEX of GC-resistant lymphocytes in vitro and in vivo, but shows significant hepatotoxicity in vivo. A significantly elevated expression of Akt2 not Akt1 in intrinsically, secondarily GC-resistant lymphocytes and relapsed/refractory ALL patients implicates a more specific target for GC resistance. Mechanistically, Akt2 has a stronger binding capacity with FoxO3a compared to Akt1, and acts as a direct and major negative regulator of FoxO3a activity driving GC resistance. Pharmacologic inhibition of Akt2 more effectively restores sensitivity to GCs than inhibition of Akt1 in vitro, shows higher synergistic effect acting with DEX, and reverses GC resistance in GC-resistant T- or B- lymphoid tumors in vivo with reduced liver toxicity. In summary, these results suggest that Akt2 might serve as a more direct and specific kinase mediating GC resistance through FoxO3a/Bim signaling pathway, and Akt2 inhibition may be explored as a promising target for treating GC-resistant hematopoietic malignancies.</jats:p>	[ { "section_content": "Glucocorticoids (GCs) are widely used drugs in the treatment of lymphoid tumors as a result of their ability to induce apoptosis in lymphoid progenitor cells.A major obstacle in GC therapy, however, is the gradual acquisition of apoptotic resistance in malignant hematopoietic cells repeatedly treated with these hormones.Previous reports indicate that between 15 and 30% of pediatric acute lymphoblastic leukemia (ALL) samples are resistant to GCs 1,2 , while in refractory childhood ALL, the prevalence of GC resistance is as high as 70% 3 .A poor response to prednisone after seven days of treatment is also a strong indicator of an increased risk of relapse and therapeutic failure in pediatric ALL 1,2 .Therefore, significant efforts are underway to develop novel strategies for resensitizing GC-resistant cells to GC therapy. Mechanisms involved in GC resistance of hematopoietic tumors have yet to be elucidated, resulting in obstacles to the discovery of efficient approaches or treatments.Various FoxO transcription factors, especially FoxO3a, have been shown to regulate apoptosis in lymphocytes 4,5 .Indeed, the FoxO3a transcription factor is upregulated by GCs in 697 pre-B ALL cells 6 .Our previous study has also shown that FoxO3a plays an important role in GC-induced apoptosis of lymphocytes and sensitivity to dexamethasone (DEX) correlates negatively with expression of phosphorylated-(p-) FoxO3a 7 .A common mechanism of inactivation of FoxO transcription factors is directly phosphorylated by Akt 8 .Inhibition of Akt kinase with MK2206 enhances GC-induced apoptosis in T-ALL cell lines 9 .Grade 3 or 4 hematologic toxicities [10][11][12] and common hepatic toxicities 10 with increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of Akt inhibitors have been reported in the treatment of solid tumors in humans, however, partially limit their clinical applicability. There are two closely related, highly conserved homologs of Akt: Akt-1 and -2, each containing a PH region and a kinase domain [13][14][15] .There are obvious differences in enzyme function between Akt1 and Akt2.Akt1 is ubiquitously expressed and plays an important role in cell proliferation 16,17 while Akt2 is expressed at high levels in skeletal muscle, in the β-islet cells of the pancreas and in brown fat and is involved in the regulation of blood sugar [16][17][18] .Fillmore et al. 19 examined the expression of Akt1 and Akt2 in a variety of hematopoietic cell lines and found that the expression of Akt2 differed more than the expression of Akt1 in these hematopoietic cell lines.In human lens epithelial cells (HLECs) Akt2 is an essential kinase in counteracting oxidative-stressinduced apoptosis through promoting phosphorylation of FoxO3a and thus downregulating Bim expression 20 .The Akt2/FoxO3a/Bim pathway has been extensively studied in HLECs 20 .Therefore, in our current study, we examined the potential role of Akt isoforms Akt1 and Akt2 in the mechanism of GC resistance and explored an effective drug with less toxicity, as an option for treatment of GC-resistant hematopoietic malignancies. ", "section_name": "Introduction", "section_num": null }, { "section_content": "Aberrant activation of Akt/FoxO3a/Bim signaling pathway may be a mechanism of GC resistance in lymphoid tumor cells Unphosphorylated FoxO3a can be upregulated by DEX treatment and then translocate into nucleus and induce apoptosis in lymphocytes 7 .To examine the importance of the Akt/FoxO3a pathway in GC-induced apoptosis of lymphoid tumors we utilized CCRF-CEM cells, which are a moderately steroid-resistant cell line 21,22 .Increasing the concentration of DEX resulted in increased apoptosis of CCRF-CEM cells (Fig. 1a).Both the total p-Akt and p-FoxO3a levels, as well as the ratios of p-Akt (Ser473) to total Akt and p-FoxO3a (Ser253) to FoxO3a, decreased; the total FoxO3a expression increased (Fig. 1b).These results suggest that Akt is the major regulatory kinase that phosphorylates FoxO3a into an inactivated form and that upregulation of FoxO3a may be an indispensable process in GC-induced apoptosis.Treatment of CCRF-CEM cells with a specific concentration of DEX over multiple passages resulted in the generation of a highly resistant cell line, designated CEM-DR (Fig. 1c).In CEM-DR cell lines, the expressions of Akt and p-FoxO3a (Ser253) increased when compared with CCRF-CEM.Treatment of CCRF-CEM cells with DEX resulted in decreased p-FoxO3a (Ser253) expression, increased expressions of total FoxO3a and the pro-apoptotic protein Bim.While in CEM-DR cells, there were no significant changes observed in these proteins with a concentration of 5 µM of DEX treatment (Fig. 1d).This finding suggests that the aberrant activation of the Akt pathway may be one of the mechanisms of GC resistance in lymphocytes. ", "section_name": "Results", "section_num": null }, { "section_content": "To examine the therapeutic role of Akt inhibitors in the treatment of GC-resistant leukemias, we treated CCRF-CEM cells with DEX and an Akt inhibitor, Akt inhibitor IV.Analysis using flow cytometry shows that treatment of CCRF-CEM lymphoblasts with Akt inhibitor IV effectively restores GC-induced apoptosis and reverses GC resistance in vitro and found that 1 µM Akt inhibitor IV with 1 µM DEX is the most effective combination (Fig. 2a).The PI3K inhibitor LY294002 can also increase sensitization of CCRF-CEM cells to DEX, but with a less significant effect than the Akt inhibitor (Fig. 2b,c).Treatment with these inhibitors results in decreased expression of p-FoxO3a and increased Bim expression (Fig. 2d).Additional pathways affect GC-induced apoptosis in lymphocytes.We selected the glycolysis inhibitor 2-DG to measure its effect on tumor cell apoptosis.Prednisolone resistance is associated with an increased glucose consumption, and the use of 2-DG sensitizes prednisolone-resistant ALL cell lines to GCs 21 .We found that 2-DG sensitizes CCRF-CEM cells to DEX and results in increased expression of FoxO3a and Bim and decreased expression of p-FoxO3a (Fig. 2e,f).It was previously reported that NOTCH1 activating mutations occur in >50% of cases of human T-ALL 23 .Notch signaling was shown to antagonize GC-induced apoptosis in T-lymphocytes 24 .In CCRF-CEM cells, the Notch signaling inhibitor dapt increases apoptosis and decreases p-FoxO3a expression in the presence of DEX (Fig. 2g,h).Serum/glucocorticoid-regulated kinases (SGKs) are activated by the phosphoinositide-3 kinase (PI3K) and translocate to the nucleus upon growth factor stimulation 23 .While there are no reports regarding the relationship between SGKs and GC resistance, Náray-Fejes-Tóth and colleagues found that SGK mRNA levels in lymphocytes are markedly induced by GCs 25 .We, therefore, tested the GC-sensitizing effects of the SGK inhibitor GSK in T and B lymphocytes.Our results show that the SGK inhibitor could not restore sensitivity to DEX either in resistant T-cell or B-cell tumors (Fig. 2i). A comparison of the aforementioned pathway inhibitors with regards to their efficiency in restoring sensitivity to DEX in CCRF-CEM cells shows that the Akt inhibitor was most effective at inducing apoptosis in the presence of DEX (Fig. 2j).At a concentration of 1 µM, the Akt inhibitor combined with DEX restores sensitivity to DEX in the GC-resistant Molt-4 and Jurkat T-ALL cell lines as well as the resistant B-cell tumors SP2/0 and Raji cell lines (Fig. 2k). ", "section_name": "Inhibition of Akt leads to a greater increase in sensitivity to GCs than inhibition of other pathway", "section_num": null }, { "section_content": "We implanted CCRF-CEM cells subcutaneously into nude mice to induce tumor formation.Animals with similar tumor volumes were then injected intraperitoneally with saline, Akt inhibitor IV, DEX or Akt inhibitor IV combined with DEX for 7 days.We found that Akt inhibitor IV combined with DEX could significantly minimize the tumors (Fig. 3a,b) and prolong the survival time of the mice (Fig. 3c).We also found this therapy significantly increased GC-induced apoptosis of tumor cells and splenocytes in tumor-bearing mice (Fig. 3d,e), while Akt inhibitor IV or DEX alone did not achieve these results.Akt signaling pathways are widely distributed in cells, playing an important role in the growth, differentiation, and proliferation of cells 26,27 .To further explore effects of inhibition of Akt on liver cells, we examined the liver enzymes ALT and AST in peripheral blood of mice treated with the Akt inhibitor.Compared with the saline treated group, Akt inhibitortreated mice show severe liver damage with ALT and AST levels increasing significantly (Fig. 3f). Overexpression of Akt2, as a major negative regulator of the FoxO3a activity, mediates GC resistance in lymphocytes Our preliminary results have shown that CCRF-CEM cells are relatively sensitive to GCs; CEM-DR cells derived from CCRF-CEM cells are highly resistant to GCs after DEX stimulation; Jurkat and Daudi are intrinsically highly resistant to GCs.In addition to these cells, we selected a human liver cell line, L-02, which is considered as a natural GC-resistant cell line without the effect of GCinduced apoptosis.To explore whether Akt1 or Akt2 expression is related to GC resistance, we examined Akt1 and Akt2 expression in the aforementioned cell lines.Western blot analysis shows that Akt1 expression in all five cell lines is similar.Akt2 expression in CEM-DR, Jurkat, Daudi, and L-02 cells is significantly higher than in CCRF-CEM cells (Fig. 4a).Then we used the CCK-8 assay to determine the degree of resistance to DEX of CCRF-CEM cells at different stages of resistance construction process.Along with the half maximal inhibitory concentration (IC50) of DEX in CCRF-CEM cells increases, the expression of Akt2 increases while the expression of Akt1 did not change (Fig. 4b).To further explore if the overexpression of Akt2 is correlated with GC resistance in lymphocytes in a clinical context, we tested Akt1 and Akt2 mRNA levels in lymphoctyes from the bone marrow of 21 ALL patients, including 10 newly diagnosed patients and 11 relapsed or refractory patients (after an average of 7.2 courses of GC-containing treatment).Compared with the newly diagnosed group, the relapsed/refractory group has higher Akt2 mRNA expression while Akt1 mRNA expression did not differ significantly between the two groups (Fig. 4c).We used the receiver operating characteristic (ROC) curve to analyze if Akt2 mRNA could be an indicator of GC resistance in a clinical setting.The area under the ROC curve was 0.9818 and the optimal threshold is 16.39 with a diagnostic sensitivity of 90% and specificity of 100% (Fig. 4d). To detect if Akt2 was able to directly interact with FoxO3a, co-immunoprecipitation assay was employed.MYC-FoxO3a was co-transfected with Flag-Akt1 or Flag-Akt2 into HEK293T cells.The reaction product was first immunoprecipitated with anti-MYC antibody and then blotted with anti-MYC and anti-Akt1 or Akt2 antibodies.Western blot analysis demonstrated the more presence of Flag-AKT2 than Flag-Akt1 in MYC-FoxO3a immunoprecipitates, suggesting that AKT2 as the major regulator directly interacting with FoxO3a (Fig. 4e). Reciprocal immunoprecipitation experiments confirmed the closer association between Flag-Akt2 and MYC-FoxO3a (Fig. 4f).Moreover, immunoprecipitation of FoxO3a protein complexes from the Jurkat T-ALL cell line demonstrated that endogenous FoxO3a can interact closer with Akt2 than with Akt1 in T-ALL lymphoblast cells (Fig. 4g). Next, we used siRNA to downregulate Akt1 or Akt2 expression in GC-resistant Jurkat cells (Fig. 4h).We found that GC-induced apoptosis increased significantly after downregulation of Akt2 expression, along with the expression of p-FoxO3a (Ser253) decreased.Furthermore, the level of GC-induced apoptosis and the expression of p-FoxO3a (Ser253) did not change after downregulation of Akt1 expression (Fig. 4i,j).Then we transfected Jurkat cells with plasmid constructs increasing the expression of Akt1 and Akt2 (Fig. 4k), and also found that GC-induced apoptosis significantly decreased and the expression of p-FoxO3a (Ser253) increased after upregulation of Akt2 expression, while GC-induced apoptosis and the expression of p-FoxO3a did not change after upregulation of Akt1 expression (Fig. 4l,m). ", "section_name": "An inhibitor of Akt resensitizes tumors to GC in vivo but is associated with significant liver toxicity", "section_num": null }, { "section_content": "To examine the therapeutic role of Akt isoform specific inhibitors in the treatment of GC-resistant lymphoid tumor cells, we treated CCRF-CEM cells with DEX and A-674563 (Akt1 inhibitor), CCT128930 (Akt2 inhibitor), or Akti1/2 (Akt1/2 inhibitor).When exploring the appropriate dose of Akt isoform inhibitors with DEX in CCRF-CEM cells, we found that a concentration of 0.8 µM is the most effective dose of Akt isoform inhibitors in the presence of 0.1 µM DEX (Fig. 5a).In combination with varying concentrations of DEX from 0 to 500 nM, the Akt isoform inhibitors significantly decrease the viability of CCRF-CEM cells (Fig. 5b).Similar result was obtained in the highly GC-resistant CEM-DR cell line (Fig. 5c).Flow cytometry experiments demonstrate inhibition of Akt1 or Akt2 in CCRF-CEM, CEM-DR, Jurkat, and highly GC-resistant Burkitt's lymphoma cell line Daudi with a concentration of 0.8 µM Akt isoform inhibitors effectively restores GC-induced apoptosis.Remarkably, in Jurkat cells, Akt2 inhibitor or Akt1/2 inhibitor shows higher GC-induced apoptosis than Akt1 inhibitor (Fig. 5d).To further test the GC-sensitizing effects of Akt isoform inhibitors, we used the CCK-8 assay to test the viability of aforementioned cell lines following treatment with a fixed concentration ratio of DEX and each Akt isoform inhibitor (Fig. 5e-h).The IC50 values of DEX in aforementioned cell lines demonstrate that Akt isoform inhibitors could effectively restore sensitive to GC, especially in T-cell tumor cell lines and Akt2 inhibitor has more effective GC-sensitizing effects than Akt1 inhibitor with a lower IC50 (Fig. 5i and Table 1).Next, we used the CompuSyn method 28 to calculate the combination index (CI) to further determine the GC-sensitizing effects of the combinations (Table 1).Our analysis shows that each of the Akt isoform inhibitors act synergistically with DEX to increase apoptosis in the T-cell tumor cells.Synergy of DEX with the Akt2 inhibitor is superior to that of DEX and the Akt1 inhibitor, especially in the Jurkat cell line.While in GC-resistant B-cell line Daudi, there is no apparent synergy between DEX and the Akt isoform inhibitors. ", "section_name": "Inhibition of Akt2 more significantly enhances sensitivity to GCs than inhibition of Akt1 in T-cell tumors in vitro", "section_num": null }, { "section_content": "Increases in p-Akt1 expression are observed by western blot analysis following incubation with the Akt2 inhibitor, while p-Akt2 expression increases after incubation with the Akt1 or Akt2 inhibitor (Fig. 6a).When we tested the expression of FoxO3a/Bim signaling proteins in CCRF-CEM cells, there were no differences between the DEX treatment group and the Akt isoform inhibitors plus DEX groups in terms of expression of FoxO3a (Fig. 6b).The Akt2 or Akt1/2 inhibitor combined with DEX could significantly decrease the expression of p-FoxO3a (Ser253) and increase the expression of the pro-apoptotic protein Bim (Fig. 6c,d).The Akt1 inhibitor combined with DEX did not alter the expression levels of p-FoxO3a (Ser253) and Bim when compared to DEX alone (Fig. 6c,d). ", "section_name": "Inhibition of Akt2 resensitizes GC-resistant cells by enhancing FoxO3a/Bim signaling pathways in lymphocyte", "section_num": null }, { "section_content": "Using a subcutaneous xenograft model of CCRF-CEM cells in nude mice, we analyzed the antitumor effects of Akt isoform inhibitors combined with DEX.Animals harboring homogeneous tumor burdens were treated with normal saline (negative control, NC), DEX or Akt isoform inhibitors plus DEX for 7 days.In this experiment, treatment with the Akt2 or Akt1/2 inhibitor plus DEX shows a significant anti-leukemic effect with reduced tumor size and prolonged overall survival of mice when compared with DEX alone, while treatment with the Akt1 inhibitor plus DEX did not achieve these results (Fig. 7a,b,d).Only the combination of the Akt2 inhibitor plus DEX shows decreased spleen size when compared to the DEX alone group (Fig. 7c).The tumors highly expressing Ki-67 show large areas of necrosis when treated with DEX plus each of the Akt isoform inhibitors (Fig. 7e,f).In spleens of tumor-bearing mice, treatment with DEX alone significantly reduces human leukemia cells highly expressing CD3 and TdT.The effect is slightly weaker than the combination of DEX with each of Akt isoform inhibitors (Supplementary Fig. S1B,C). To further test the therapeutic role of Akt isoform inhibition in GC-resistant B lymphocytes in vivo, we established an allograft model of SP2/0 cell line, a GCresistant myeloma cell line of mouse.In this experiment, animals treated with DEX combined with Akt2 or Akt1/2 inhibitor show significantly reduced tumor size similar to those observed in CCRF-CEM xenograft model (Fig. 7g,h).Akt isoform inhibitors significantly increase the GCinduced apoptosis of tumor cells in mice (Fig. 7i).Only treatment with Akt1/2 inhibitor plus DEX decreases spleen size when compared to the DEX alone group (Fig. 7j).Seven days of treatment of Akt isoform inhibitors plus DEX could not prolong the overall survival of tumorbearing mice (Fig. 7k).When the time of treatment extended to 11 days, Akt2 inhibitor plus DEX prolong the overall survival of mice (Fig. 7l). ", "section_name": "Inhibition of Akt2 reverses GC resistance in vivo", "section_num": null }, { "section_content": "When exploring the influence on liver cell's viability of Akt1, Akt2, or Akt1/2 inhibitors in vitro, we measured L- 02 cell viability following a 6, 12, 24, 36, or 48 h incubation with each inhibitor.Our data shows the influence on liver cell's viability by Akt2 inhibitor is minimal: cell viability in this group is gradually restored and by 24 h always higher than that in Akt1 and Akt1/2 inhibitor groups (Fig. 8a,b).To further test the damage to liver by Akt isoform inhibitors in vivo, we found that the Akt1 inhibitor leads to higher AST and total bilirubin (TBIL) levels, the Akt1/2 inhibitor leads to increased ALT, AST, and TBIL levels, while the Akt2 inhibitor did not show significantly increased liver function indices when compared with negative control group, indicating that inhibition of Akt2 has milder liver toxicity in vivo (Fig. 8c-e).Western blot analysis demonstrate that the Akt isoform inhibitors also enhance the FoxO3a/Bim signaling pathway in liver cell by increasing FoxO3a expression, lowering p-Foxo3a expression and increasing Bim expression (Supplementary Fig. S2). When exploring the influence on other viscera functions by Akt isoform inhibitors in vivo, we found that Akt isoform inhibitors did not change white blood cell (WBC) count, platelet (PLT) count, blood glucose or creatinine (Cr) level of nude mice (Supplementary Fig. 8F, I-K).Due to the DEX stimulation, red blood cells (RBCs) from the reserve pool transfer to the circular pool then RBC count and hemoglobin (HGB) levels increase (Supplementary Fig. 8G,H).We also examined the pathological changes of Akt isoform inhibitors in vital organs and found that Akt isoform inhibitors did not cause the morphological changes in heart, kidneys, liver, or lungs of mice (Supplementary Fig. S3). ", "section_name": "Inhibition of Akt2 shows less liver toxicity than inhibition of Akt1", "section_num": null }, { "section_content": "GCs are common components in many chemotherapeutic protocols for lymphoid malignancies.Although they are effective in the initial stages, patients often develop resistance to GCs on relapse.Recently, some studies have proposed that the molecular mechanisms of resistance to GCs in ALL may be associated with RASpathway activating mutations 29,30 and NALP3 inflammasome upregulation and CASP1 cleavage of the GC receptor 31 .Li et al. 32 performed genome sequencing on pre-treatment and post-treatment samples from 82 pediatric T-ALL patients, and found that mutations in IL-7R signaling pathway genes including JAK1, KRAS, IL-7R, JAK3, NF1, NRAS, and Akt were associated with both GC resistance and poor outcomes.Therapeutic targeting of the IL-7Ra signaling pathways including the Akt inhibitors were also recommended in ALL treatment 33 , but the cellular mechanisms of Akt signaling pathway underlying GC resistance were not fully elucidated in this study. In our current study, we chose inhibitors of the PI3K pathway, Akt pathway, glycolysis pathway, NOTCH1 pathway and SGKs pathway, which have been reported to A combination index (CI) below 1.0 indicates a synergistic interaction, equal to 1.0 indicates an additive interaction and greater than 1 indicates an antagonistic interaction 28 affect GC-induced apoptosis in lymphocytes 21,[23][24][25] to test their effects on GC-sensitization.We found all these pathway inhibitors enhance the FoxO3a/Bim pathway to reverse GC resistance, which further confirms that the FoxO3a/Bim pathway is essential for the development of GC resistance in lymphocytes.Through the analysis of the role of Akt isoforms Akt1 and Akt2 in the GC resistance mechanism, we found Akt2 is the major kinase phosphorylating FoxO3a and the expression of Akt2 in vitro or vivo correlates negatively with sensitive to GCs.Selective inhibition of Akt2 enhances the FoxO3a/Bim signaling pathway and more significantly restores the sensitivity to GCs than selective inhibition of Akt1.According to our result that Akt2 expression increases in GC-resistant lymphocytes especially secondarily GC-resistant CEM-DR cells, we further concluded that Akt2/FoxO3a/Bim signaling pathway is the mechanism of the gradual acquisition of GC resistance in lymphocytes repeatedly treated with hormones, and Akt2 not Akt1 may be the direct reversal target of GC resistance in lymphoid malignancy.Inhibition of Akt with the Akt inhibitor IV in our study was shown to have a remarkable liver toxicity and other Akt inhibitors [10][11][12] such as perifosine 10 are also reported to be associated with the common hematologic and hepatic toxicities in clinical, negatively affecting their prospects for clinical applications.When testing the toxicity of Akt isoform inhibitors, we found that the Akt2 inhibitor has milder liver toxicity than the Akt1 or Akt1/2 inhibitors.This may be explained by the obvious differences in enzyme function of Akt1 and Akt2: Akt1 is ubiquitously expressed and plays an important role in cell proliferation 16,17 while Akt2 is expressed at high levels in skeletal muscle and brown fat and is involved in the regulation of blood sugar [16][17][18] .Inhibition of Akt1 results in suppression of downstream proliferation pathways such as the mTOR pathway, especially in some eugonic cells such as liver cells and bone marrow cells; while inhibition of Akt2 may have more influence on the glucose metabolism and may be correlated with the reported hyperglycemia of Akt inhibitors in clinical [10][11][12] . As inhibition of Akt2 can effectively reverse GC resistance, they may represent a novel and promising treatment to overcome GC resistance in lymphoid tumors.An Akt2 inhibitor combined with GCs may be used in the future for treatment of GC-resistant lymphoid tumors. ", "section_name": "Discussion", "section_num": null }, { "section_content": "", "section_name": "Materials and methods", "section_num": null }, { "section_content": "The phosphoinositide-3 kinase (PI3K) inhibitor LY294002, the glycolysis inhibitor 2-deoxy-D-glucose (2- ", "section_name": "Inhibitors and drugs", "section_num": null }, { "section_content": "The mouse lymphocytic leukemia cell line L1210, the human acute T-lymphoblastic leukemia cell line CCRF-CEM, the human acute T-cell leukemia cell line Molt-4, ", "section_name": "Cell lines", "section_num": null }, { "section_content": "Twenty-one ALL samples from 10 newly diagnosed patients and 11 relapsed/refractory patients (after an average of 7.2 courses of GC-containing treatment) were selected.Approval for the study was obtained from the Human Research Committee of Huadong Hospital Affiliated to Fudan University, and informed consent was obtained in accordance with the Declaration of Helsinki. ", "section_name": "Patient samples", "section_num": null }, { "section_content": "HEK293T cells grown at 80% confluency were transfected with Turbofect.Cells were co-transfected with plasmid vectors containing MYC-FoxO3a and Flag-Akt1/ Flag-Akt2.Single transfection with one of the plasmids alone was served as control.Twenty-four hours later, cells were lysed on ice by using 500 μl RIPA lysis buffer.The detergent soluble fraction was recovered by centrifugation at 4 °C for 20 min at 12,000 r/min and supernatants were subjected to immunoprecipitation with mouse anti-MYC or anti-Flag antibody.Immune complexes were isolated with protein G PLUS-Agarose beads.The immunoprecipitated products were washed four times with lysis buffer, eluted with 2 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer and analyzed by western blotting. ", "section_name": "Co-immunoprecipitation assay", "section_num": null }, { "section_content": "Short-interfering RNA (siRNA) targeting Akt1 and Akt2 were prepared by GenePharma corp.The sequences of siRNA targeting Akt1 were 5ʹ-GGCCCAACACCUUCAU-CAUTT-3ʹ(sense) and 5ʹ -AUGAUGAAGGUGUUGGG CCTT-3ʹ(anti-sense).The sequences of siRNA targeting Akt2 were 5ʹ-GGUUCUUCCUCAGCAUCAATT-3ʹ(sense) and 5ʹ-UUGAUGCUGAGGAAGAACCTT-3ʹ(anti-sense).Scrambled siRNA was used as a control.Transfection of siRNAs was done according to the manufacturer's protocol.The transfection efficiency of siRNA in Jurkat cells was more than 50% by flow cytometry detection. ", "section_name": "Short-interfering RNA transfection", "section_num": null }, { "section_content": "Enhanced green fluorescent protein (EGFP)-tagged plasmid constructs targeting Akt1 and Akt2 were prepared by GeneCopoeia corp.The plasmid of retroviral vector p-LXSN-Aktl, which contained the whole Aktl gene sequence, and p-LXSN-Akt2, which contained the whole Akt2 gene sequence, were used to transfect Jurkat cells with fuGENE according to the manufacturer's protocol.The empty vector p-LXSN was used as a control.And then the positive cell clones were selected with antibiotics G418.The transfection efficiency of retroviral vector in Jurkat cells was 30-40% by flow cytometry detection. ", "section_name": "EGFP-tagged plasmid constructs of retroviral vector transfection", "section_num": null }, { "section_content": "The animal protocol was approved by the institutional animal use committee of the Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences).Female nude mice (4-5 weeks of age) were obtained from the animal department of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, and were maintained in pathogen-free conditions.Human acute leukemia cells CCRF-CEM or mouse myeloma cells SP2/0 were implanted in the armpit of 4-5-weeks-old female nude mice with the number of 1 × 10 7 cells per mouse, which could grow into tumors by day 35.When the tumor size reached 500 mm 3 , all the mice received different treatments administered intraperitoneally.Based on in vitro optimization, inhibitor doses were as follows: the Akt isoform inhibitors plus DEX group received 2 × 10 -3 µmol Akt isoform inhibitor and 0.1 mg DEX per mouse, the DEX group received 0.1 mg DEX per mouse, and the negative control group received only saline.Akt isoform inhibitors and DEX were administered on days 1-7 or days 1-11. ", "section_name": "Establishment of tumors in mice", "section_num": null }, { "section_content": "Single-cell suspensions from tumors and spleen were prepared essentially by enzymatic digestion.Resected tumors and spleen were minced into small (1-2 mm 3 ) pieces with a scalpel, and immersed in 10 ml of digestion mixture ((phosphate buffer saline) PBS, 0.5 mg/ml collagenase A, 0.2 mg/ml hyaluronidase and 0.02 mg/ml DNase I) per 0.25 g of tumor or spleen tissue.This mixture was incubated at 37 °C for 45 min on a rotating platform.The resulting cell suspensions were filtered sequentially through 60-and 40-µm cell strainers and washed with PBS.Then single-cell suspensions with cell concentration of 10 6 /ml were stained with FITC-labeled anti-annexin-V and/or prodium Iodide antibody (Sigma-Aldrich, USA), and analyzed by flow cytometry (BD Corp., USA). ", "section_name": "Preparation of single-cell suspensions from tumors or spleen and flow cytometric analysis of cell apoptosis", "section_num": null }, { "section_content": "We performed statistical analysis using Student's t-test.We considered results with p < 0.05 as statistically significant.Survival in animal experiments was represented with Kaplan-Meier curves and significance was estimated with the log-rank test. ", "section_name": "Statistical analyses", "section_num": null } ]	[ { "section_content": "Acknowledgements This work was supported by Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Laboratory Animal Center, College of Pharmacy, Fudan University.We are grateful to Yongping Xu for generation of T-ALL xenografts, Qi Dang for help in RNA transfection procedures, Yanyun He for help in flow cytometer detection, and Qi Jiang for help in animal procedures and revision of figures.This work was supported by grants from the Fujian Provincial Health and Family Planning Youth Scientific Research Project [2016-1-54]. ", "section_name": "", "section_num": "" }, { "section_content": "Author details 1 Department of Hematology & Shanghai Key Laboratory of Clinical Geriatric Medicine, Huadong Hospital Affiliated to Fudan University, Shanghai 200040, China. 2 Senior Department of Hematology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China. 3The School of Life Sciences, Shanghai University, Shanghai 200444, China.Authors' contributions M.X. and A.Y.: conception and design of the study, experimental studies, data acquisition, analysis and interpretation of data, drafting the article; J.M. and K.W.: experimental studies, statistical analysis; M.W. and H.X.: data acquisition, statistical analysis; Y.J.: conception and design of the study, manuscript editing and revision; Y.X.: conception and design of the study, analysis and interpretation of data and manuscript editing and revision.All authors approved the final version of the article to be published. The authors declare that they have no conflict of interest. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Information accompanies this paper at (https://doi.org/10.1038/s41419-018-1043-6). ", "section_name": "Conflict of interest", "section_num": null }, { "section_content": "Author details 1 Department of Hematology & Shanghai Key Laboratory of Clinical Geriatric Medicine, Huadong Hospital Affiliated to Fudan University, Shanghai 200040, China. 2 Senior Department of Hematology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China. 3The School of Life Sciences, Shanghai University, Shanghai 200444, China.Authors' contributions M.X. and A.Y.: conception and design of the study, experimental studies, data acquisition, analysis and interpretation of data, drafting the article; J.M. and K.W.: experimental studies, statistical analysis; M.W. and H.X.: data acquisition, statistical analysis; Y.J.: conception and design of the study, manuscript editing and revision; Y.X.: conception and design of the study, analysis and interpretation of data and manuscript editing and revision.All authors approved the final version of the article to be published. ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare that they have no conflict of interest. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Information accompanies this paper at (https://doi.org/10.1038/s41419-018-1043-6). ", "section_name": "Conflict of interest", "section_num": null } ]
10.3390/diagnostics13050964	Perspectives on the Application of Cytogenomic Approaches in Chronic Lymphocytic Leukaemia	<jats:p>Chronic lymphocytic leukaemia (CLL) is a haematological malignancy characterised by the accumulation of monoclonal mature B lymphocytes (positive for CD5+ and CD23+) in peripheral blood, bone marrow, and lymph nodes. Although CLL is reported to be rare in Asian countries compared to Western countries, the disease course is more aggressive in Asian countries than in their Western counterparts. It has been postulated that this is due to genetic variants between populations. Various cytogenomic methods, either of the traditional type (conventional cytogenetics or fluorescence in situ hybridisation (FISH)) or using more advanced technology such as DNA microarrays, next generation sequencing (NGS), or genome wide association studies (GWAS), were used to detect chromosomal aberrations in CLL. Up until now, conventional cytogenetic analysis remained the gold standard in diagnosing chromosomal abnormality in haematological malignancy including CLL, even though it is tedious and time-consuming. In concordance with technological advancement, DNA microarrays are gaining popularity among clinicians as they are faster and better able to accurately diagnose the presence of chromosomal abnormalities. However, every technology has challenges to overcome. In this review, CLL and its genetic abnormalities will be discussed, as well as the application of microarray technology as a diagnostic platform.</jats:p>	[ { "section_content": "Chronic lymphocytic leukaemia (CLL) is a chronic lymphoproliferative disorder characterised by accumulation of mature monoclonal B lymphocytes, more than 5000 per microlitre in peripheral blood, positive for immunophenotype marker (CD5+ and CD23+) and/or the involvement of lymph nodes [1].It is a common type of leukaemia in adults, especially in Western countries.The estimated incidence of this disease in the Western population (USA and Europe) is approximately 5 new cases per 100,000 individuals, regardless of gender [2].In the USA itself, the estimated number of newly diagnosed cases for 2020 was 21,040 cases, which was around 1.2% of all cancer cases.The median age at diagnosis of this disease is 72 years old [3][4][5]; there is male predominance with a male-to-female ratio of approximately 2:1 [6][7][8].It accounts for about 1% to 3% from total non-Hodgkin lymphoma cases reported.In contrast, the CLL cases reported in Asian countries as well as East Asia (0.1-0.2/100,000) [9,10], Africa (0.66/100,000) [11], and South America (Hispanic descendants) (1.17/100,000) [12] are relatively low compared to their Western counterparts [13][14][15][16][17][18][19][20].Further, in Japan, CLL is classified as a rare disease, with the reported incidence rate being far below 0.5 per 100,000 person-years [21][22][23][24].It is challenging to diagnose CLL in Japan due to the disease's high degree of morphological and immunological variability [25].For Australia and New Zealand, CLL is considered a common type of leukaemia to be diagnosed with, having an incidence rate of 2.99 per 100,000 [26].In contrast, CLL is a rare disease in Africa [27,28]. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "CLL is a heterogeneous disease.Its pathogenesis can be viewed as cooperation between a patient's risk factors and genetic aberrations.There have been several studies performed to identify risk factors for CLL development; however, to date, there is still no specific acquired factor that has been identified for disease development.However, there is strong evidence that genetic predisposition can lead to CLL [36][37][38][39].Host factors including family history with haematological malignancy (CLL and/or non-Hodgkin lymphoma (NHL)) are among the strong evidence that has been studied.The study performed by Slager et al. revealed that relatives of CLL patients have a 2-to 8-fold increase in the risk of developing CLL and a 2-fold increased risk of getting NHL compared to the general population [40].This finding was also supported by those of Goldin et.al, which state that familial CLL was diagnosed at an earlier age compared to sporadic CLL [41].There are also case reports involving familial CLL where two or more individuals were affected by CLL in the same family. Histone modifications, such as those linked to active enhancer and promoter elements and regions of the genome that were actively transcribed, have been shown to play a role in the epigenetics of CLL.Additionally, it has been discovered that single-nucleotide polymorphisms (SNP), which increase the risk of CLL, overexpress transcription factor binding [42].The latest studies using genome-wide association studies (GWAS) revealed more than 40 susceptibility loci which were important in B lymphocytes and apoptotic pathways [42,43]. The common chromosomal aberrations associated with CLL are del 13q14, trisomy 12, del 11p, and del 17p [44][45][46][47].Other chromosomal aberrations observed in CLL are deletions in 6q, 9p21, and 10q23, total or partial trisomy of chromosomes 3, 8, 18, or 19, and duplications in 2p24 [47][48][49][50].The most common genetic lesions in CLL are deletions of 13q14 (del 13q14), generally monoallelic in 50~60% patients (Figure 1; Table 1) [18], and involve the deletion of regions containing two long non-coding RNA genes (DLEU2 and DLEU1) which later develop clonal lymphoproliferation, recapitulating the different steps of CLL initiation and progression.Deletion 13q14 causes dysregulation of microRNAs, i.e., miR15A and miRNA16A, which are encoded in the deleted region.Both microRNAs have critical roles in controlling the production of proteins essential for cell apoptosis and normal cell cycle progression [51].Consequently, cells are unable to respond to stress signals in a way that promotes apoptosis and leads to disease progression when these microRNA regions are absent [52].Besides that, deletion of miR16A and miR15A causes upregulation of the BCL2 gene in CD5+ cells, which activates the BCL-2 proto-oncogene aberrant signalling pathway and assist in the development of the disease [53].Deletion 13q14 is associated with good prognosis as well as prolonged time to first treatment (TTFT) and prolonged overall survival compared to other genetic abnormalities [44,51]. Deletion of 17p, especially at the region 17p13 chromosomal region (del17p), can be found at different frequencies depending on clinical stages of CLL disease, ranging from 1%-3% during initial diagnosis to 20% in chemo-refractory disease (Figure 1; Table 1) [48,54].Deletion 17p is associated with TP53 inactivation, thus causing genomic instability.This deletion is also linked to resistance to DNA-damaging agents (radiotherapy or chemotherapy) and presence at diagnosis usually indicates unfavourable OS and decreased TTFT.Trisomy 12 is a chromosomal aberration in CLL found in 10-20% of cases and often appears as a unique cytogenetic alteration (40-60% of cases with trisomy 12) (Figure 1; Table 1).In addition, it can be associated with other chromosomal aberrations, including trisomy 18 and 19, recurrent CLL deletions (e.g., 14q, 13q, 11q, or 17p), and IGH translocations [54].Trisomy 12 is also associated with an atypical morphology of the lymphocytes.Although trisomy 12 is considered an intermediate-risk genetic lesion in CLL, the co-occurrence with NOTCH1 mutations are associated with poor survival outcome [55].This finding is also in line with the increased frequency of trisomy 12 in Richter syndrome patients. Deletion of the long arm of chromosome 11 is detected in 5-20% of CLL patients (Figure 1; Table 1) [45,56,57].This deleted region of chromosome 11 usually harbours ATM gene in almost all cases, as well as other genes including RDX, FRDX1, RAB39, CUL5, ACAT, NPAT, KDELC2, EXPH2, MRE11, H2AX, and BIRC3.ATM gene mutations have been largely studied in CLL patients with del(11q); however, they have been found in only 8-30% of 11q-patients [58,59], indicating that other genes could play a role in the pathogenesis of 11q deletions in CLL.One of these genes is BIRC3, which is located near to the ATM gene, at 11q22.BIRC3-disrupting mutations and deletions have been rarely detected in CLL at diagnosis (4%) but are detected in 24% of fludarabine-refractory CLL patients, suggesting that BIRC3 genetic lesions are specifically associated with a chemorefractory CLL phenotype [60,61].CLL patients with del(11q) are characterised by large and multiple lymphadenopathies and have been associated with progressive disease and poor prognostic factors, such as unmutated IGHV genes.It has been associated with shorter TTFT, shorter remission durations, and shorter OS following standard chemotherapy compared to non-deleted 11q (and non-deleted 17p) cases [62]. Deletion of 17p, especially at the region 17p13 chromosomal region (del17p), can be found at different frequencies depending on clinical stages of CLL disease, ranging from 1-3% during initial diagnosis to 20% in chemo-refractory disease (Figure 1; Table 1) [48,54].Deletion 17p is associated with TP53 inactivation, thus causing genomic instability.This deletion is also linked to resistance to DNA-damaging agents (radiotherapy or chemotherapy) and presence at diagnosis usually indicates unfavourable OS and decreased TTFT.Poor [48,54] In addition to the common chromosome aberrations detected by FISH, Table 2 displays various chromosome abnormalities in CLL patients revealed by other platforms.Robbe et al. (2022) identified 74 regions of the genome that were currently affected by copy number alterations (CNAs), including 14 well-known CNAs such as del13q14.2,del11q22.3,and del17p13.1,through microarray.Another 60 regions-of which, 27 were previously not recognised and the remaining 33 CNAs-could be refined to a smaller minimal overlapping region.The author also demonstrated the most likely target gene for nine known regions, includingTP53/del17p13.1, and seven additional regions, including PCM1/del8p, IRF2BP2/del1q42.2q42.3, and SMCHD1/del18p11.32-p11.31[72]. Certain gene mutations, in addition to chromosomal abnormalities, are critical to CLL pathogenesis, and multiple subpopulations of evolving malignant cells have been identified.These modifications have an impact on intracellular or microenvironmentdependent signalling pathways [58].Over 5% of CLL patients have mutations in NOTCH1, ATM, SF3B1, and TP53.Notch proteins regulate the development of haematopoietic cells by acting as cell transmembrane receptors.Mutations in NOTCH1 at proto-oncogenes' coding and non-coding regions can worsen disease through splicing events and increase their overall activity [73].ATM, as previously stated, is a gene that detects damaged DNA and induces cell apoptosis, and its mutation will lead to dysregulation of the cell cycle [74].SF3B1 is the gene that produces nuclear ribonucleoproteins, which are required for messenger RNA splicing and, thus, affect the cell cycle [75].As previously stated, TP53 is essential for responding to DNA damage and inducing cell apoptosis. Aberrant signalling pathways also play important roles in the pathophysiology of CLL.The three main pathways involved are antigen-independent BCR signalling, BCL2 protooncogene upregulation, and impaired DNA damage response.Through antigen-independent or antigen-dependent autonomous signalling of CLL cells, the antigen-independent BCR signalling pathway directly affects cell survival, growth, differentiation, and cellular adhesion or migration.It is influenced by low miR150 levels as well as high FOXP1 and GAB1 expression [76].BCR activation causes the kinases such as PI3K, SYN, BTK, and LYN to be activated, which results in cytoplasmic domain integrin activation and conformational changes that allow more ligand to bind to integrin's extracellular activity, affecting cell proliferation, migration, differentiation, and survival [77].Somatic mutations in immunoglobulin heavy chain variable region (IGHV) genes also affect the antigen-independent BCR signalling pathway.Mutated IGHV has weaker BCR signalling due to narrower antigen specificity, resulting in a higher mutation burden and a lower frequency of driver mutations.As a result, mutated IGHV CLL cells proliferate more slowly, making the disease process more benign and less clinically aggressive.Unmutated IGHV CLL cells, on the other hand, have sustained BCR signalling by binding to multiple epitopes, resulting in a lower mutation burden and a higher driver mutation frequency.This process eventually leads to faster clonal expansion and more clinically aggressive disease [75,78].Table 3 highlights the gene mutations that contribute to the prognosis of CLL. Even though the incidence of CLL in Western countries is higher than in Asian countries, the disease progression in Asian patients has been reported to be more aggressive and with a shorter time to treatment compared to its counterpart.This event was postulated to happen due to different biomarkers and susceptibility in Asian populations.Based on a prospective study conducted in Senegal by Sall et al., CLL was found to be more aggressive and had a poorer prognosis at a younger age than in developed nations [29].To depict the exact pattern of disease progression in African countries, however, it was necessary to conduct large-scale epidemiological research in African countries, as this study only represents a small-scale African study [13].There were several case reports showing Asian CLL had reported a few different chromosomal aberrations than Western CLL.Western and Asian CLL shared the major copy number changes, which are del13q14, trisomy 12, deletion 17p, and deletion 11q [79].Kawamata et al. also reported that Asian CLL patients more frequently have either trisomy/duplication of 3q or trisomy 18/dup18q; none of these chromosomal aberrations were reported in Western CLL patients [80].Another study performed by Wu and his team members revealed Asian CLL patient had high frequency of TP53 mutation compared to Western CLL [81,82].Prior to the last two decades, it was reported that the common chromosome abnormalities of CLL in South Africa are comparable to those of the rest of the world [83].In 2016, Sall and colleagues found that CLL patients in Senegal exhibited the same clinical presentation as individuals globally.The epidemiology of haematologic malignancies, particularly CLL, is less understood in Latin America (Central and South America) [84].A study performed by Hahn and his colleagues discovered two gene candidates, PRPF8 and SAMHD1, in Australian familial CLL [85].Even though African CLL is considered rare, their patients usually have a younger median age of onset (59 years old), higher frequency of adverse prognostic factors, and poor clinical outcome.It also found that TP53, SF3B1, and NFKBIE mutations in African CLL is higher than in Western CLL [86].For almost 40 years, the Rai and Binet clinical staging systems, which base their evaluations on a patient's physical examination as well as their blood counts, have served as the foundation for determining a patient's prognosis in CLL [87,88].Rai and modified Rai classification stress the lymphocytes count and nodal and organ (spleen) involvement more, while Binet classification looks more at haemoglobin level, platelet count, and number of nodal areas involved.However, the information gained from these classifications during diagnosis of CLL in patients will not be able to predict the progression of disease in each individual [1].Recently, an international team of researchers reviewed data from patients participating in eight randomised clinical trials in Europe and the United States in order to construct a prognostic score that contains widely available clinical, biochemical, and genetic prognostic characteristics.The CLL International Prognostic Index (CLL-IPI) was developed as a result of this international effort, and it is a reasonably straightforward prognostic tool.This prognostic model divides patients into four distinct categories, each of which has a significantly different overall survival rate, based on five parameters such as age, clinical stage, TP53 status (normal vs. del(17p), and/or TP53 mutation), IGHV mutational status, and serum β2-microglobulin.Subsequently, the prognostic utility of the CLL-IPI was validated in two separate cohorts of newly diagnosed patients, one from the Mayo Clinic and the other from the Swedish CLL registry [89].Despite the fact that CLL-IPI was initially established to predict overall survival, it was found that the index could also predict TTFT in newly diagnosed patients with CLL.Only 20% of the original dataset consisted of patients with early illness, and no effort has been made to optimise the CLL-IPI risk score to stratify TTFT among early-stage patients.It is important to emphasise that TTFT is a disease-specific goal that is more relevant than overall survival for patients who have recently been identified with early-stage disease [90][91][92].The CLL-IPI is used as a supplement for the existing methods of risk stratification for CLL [93]. ", "section_name": "Genetics of CLL", "section_num": "2." }, { "section_content": "For decades, diagnosis of CLL was performed using a full blood picture with the presence of lymphocytes more than 5 × 10 9 /µL, examination of marrow morphology, marrow immunophenotyping, marrow cytogenetics, and clinical examination to detect nodal involvement.However, for the past 10 years, rapidly developed technology has made the detection of genetic aberrations in haematological malignancies, especially in CLL, become more comprehensive and elaborate.Genetic aberration detection plays a pivotal role in diagnosis, disease prognosis determination, risk stratification, and survival outcome.It is also essential in specific targeted therapy selection that is tailored to a patient's genetic aberrations in order to achieve a better outcome [37].Various methods of cytogenomic testing can help clinicians to detect the presence of genetic aberrations in patients. Cytogenomics can be defined as the study of the numerical and structural variation of the genome at the chromosomal and subchromosomal level as well as at a molecular resolution using methods that cover the entire genome or specific DNA sequences [94,95].It evaluates chromosomes and their relation to disease [96].The term \"cytogenomics,\" also called \"chromosomics,\" was proposed by Uwe Claussen to highlight the three-dimensional morphological changes that occur in chromosomes and which are crucial aspects in the regulation of genes [97].Cytogenomic testing is not limited to conventional cytogenetic analysis (CCA) and molecular cytogenomics methods, i.e., fluorescence in situ hybridisation (FISH), polymerase chain reaction (PCR), or Multiplex Ligation-dependent Probe Amplification (MLPA); it also comprises high-throughput cytogenomics technologies which include applications of whole-genome Copy Number Variation (CNV) analysis such as DNA microarray, next-generation sequencing (NGS), and, more recently, GWAS as a diagnostic method [98,99].These fancy, sophisticated, and typically very costly methods are only possible in conjunction with high-tech apparatuses and/or bioinformatics.In return, they are competent for achieving a high-resolution view of genomes as well as the generation of massive data sets in a time-effective manner [100].Furthermore, cytogenomics exemplify the understanding of genomic instability and its association with normal and abnormal aging throughout ontogeny which later may contribute to cancer development [101]. Until now, CCA still remains the gold standard to diagnose chromosomal aberrations in CLL, especially in detecting the presence of complex karyotypes or balanced chromosomal translocations [54,102,103].However, CCA is time-consuming, unable to assess non-dividing cancer cells, and sometimes yields poor morphology or inadequate cells for analysis [104,105].It also can only detect chromosomal aberrations around 30% of CLL cases [106,107].In developed countries, this method has become the last choice as array-based testing is more favoured and CCA only acts as last resort in detecting balanced chromosomal abnormalities. Based on a number of prospective clinical trials, the latest International Workshop on Chronic Lymphocytic Leukemia (iwCLL) guidelines for the management of CLL recommend performing FISH analysis as well as analysis of the TP53 gene in all patients with CLL, in both general practice and clinical trials.The use of CCA is recommended only in the context of clinical trials rather than routine clinical settings.This recommendation is mostly based on recent reports highlighting the prognostic significance of complex karyotype (CK) which, presently, can be detected only through CCA [94,108]. In CLL, CK is classically defined as the presence of ≥3 clonal structural or numerical abnormalities.Although present in 8% of monoclonal B lymphocytosis cases, 26 CK ≥3 is associated with advanced-stage disease, cases harbouring unmutated IGHV genes (U-CLL), del(11q), TP53 aberrations [del(17p) and/or TP53 mutation], and telomere dysfunction [109,110]. Combining FISH with NGS, as well as FISH and long-range sequencing methods, has led to significant advances in the field of cytogenomics in the 2010s [111,112].FISH techniques are the most effective for researching genomes' repetitive sections [113], and as a result, numerous probes targeting heterochromatic and euchromatic areas of the human genome have been created [111].In early 2010, FISH and MLPA were becoming more popular as tools to diagnose chromosomal aberrations in CLL.However, despite the high sensitivity test for both methods, they are limited to specific known genomic loci [114,115].FISH and MLPA act as the supplementary test to CCA.Both can be used in diagnosing genetic aberrations in non-dividing cells with high specificity and sensitivity.FISH is also able to detect low levels of mosaicism and mosaics of mono-and biallelic deletions [116,117].However, FISH testing needs to be performed separately with specific probes for each genomic abnormality, making this method relatively expensive and time-consuming.It also unable to detect any other chromosomal abnormalities aside from the known genomic loci that have been specified by probes [47].FISH is more sensitive than karyotyping; nevertheless, it is only effective for analysing specified loci, and it requires an assay for each targeted aberration [118].While MLPA testing is able to detect copy number alterations, methylation pattern changes, and/or even point mutations simultaneously in multiple target regions [114,[119][120][121], it has its own disadvantages.This method cannot detect copyneutral loss of heterozygosity and has problems with mosaicism, i.e., unable to be obtained, tumour heterogeneity, or sometimes can cross contaminate with normal cells [116].This finding proves that FISH and MLPA cannot be a stand-alone test and only able to act as complementary test for CCA. The emergence of microarray-based comparative genome hybridisation (array-CGH) and high-density single-nucleotide polymorphism (SNP) arrays has led to deeper understanding of the CLL genomic landscape.By delivering a genome-wide, high-resolution analysis that does not require cell culturing or viable cells for testing, chromosomal microarray analysis fills the void between genome-wide low-resolution chromosome studies and region-limiting disease-specific targeted FISH panels [122,123].However, array-CGH has a several shortcomings, including its inability to detect low-level mosaics, its insensitivity to heterochromatin, and its inability to detect balanced aberrations.Only copy number variations were able to be identified between the years 2000 and the 2010s [124][125][126].Initially, microarray-based detection of copy number alterations (CNAs) is the standard of care for the diagnosis of most constitutional chromosomal imbalances in children with developmental disability abnormalities [123], but recently it has become more popular for diagnosing haematological malignancies.Microarray technology, especially that using CNA+SNP chip technology, is the best at diagnosing aneuploidies, microdeletions, especially cryptic loci deletion and duplications, as well as amplification in CLL.It also can detect additional confirmation of CNAs and the ability to detect copy-neutral loss of heterozygosity (CN-LOH) and some polyploidies.The integration of microarray analysis into the cytogenetic diagnosis of haematological malignancies improves patient management by providing clinicians with additional information about potentially clinically actionable genomic alterations [123].However, every technology has its own limitation.Microarray limitation include the inability to detect balanced rearrangements, decreased performance at low levels of tumour [50], the need for well-trained laboratory technologists, and high operation costs, even though this method is far superior compared to CCA, FISH, and MLPA [127]. Examples of commonly used microarray platforms in haematological malignancies are the CytoScan HD array platform (Affymetrix) and the HumanOmniExpress Array (Illumina).Both platforms use CNA+SNP chip technology in detecting cytogenomic alterations.Data obtained by the CytoScan HD array platform supplied by Affymetrix were analysed using the Chromosome Analysis Suite software while HumanOmniExpress platform data were analysed using Nexus copy number software (Biodiscovery Inc.) using annotations of genome version GRCh37 (hg19).In a study done in the Netherlands by Steven-Kroef et.al, both platforms show a high limit of resolution and detection of clinically relevant genomic aberrations which were unable to be detected by CCA and FISH [127]. For the past few years, optical genome mapping (OGM) has emerged as a promising new approach that may be able to circumvent all of the aforementioned testing hurdles with a single, comprehensive analysis.OGM is based on high-throughput imaging of long DNA molecules (>250 Kb) that have been fluorescently labelled at a specific 6 bp sequence motif found about 15 times per 100 Kb in the human genome [128].The unique labelling pattern throughout the genome allows for the unambiguous identification of every imaged molecule's genomic location, resulting in a local consensus map that can be compared to a reference genome to detect structural variants (SVs).The so-called rare variant pipeline is used for this study; it targets mosaic samples and can discover SVs from single molecules across the genome, beginning at 5 Kb and falling to a fraction of 1% in allele frequency.In addition, information on the depth of the genome's coverage is utilised in order to recognise copy number variants (CNVs) and whole-chromosome aneuploidies [129].Several recent studies have shown that OGM performs well in the cytogenomic assessment of various haematological malignancies, with a particular emphasis on myeloid neoplasms (acute myeloid leukaemia and myelodysplastic syndromes) and acute lymphoblastic leukaemia cases.In these studies, OGM was able to efficiently detect the bulk of clinically relevant abnormalities reported by standard approaches, while at the same time revealing new cytogenomic information in some situations [130,131].A cohort study done by Puiggros and her team on 46 CLL patients found that the usage of OGM in CLL enabled them to achieve better characterisation of these patients' genomic complexity in comparison to current approaches, and also showed increasing detection of cytogenomic abnormalities via the OGM approach which can contribute to adverse disease progression in those CLL patients [103]. NGS genomic oncology profiling assays and GWAS brought into play an unpreceded analytical depth to accommodate the characterisation of the highly complicated genetic landscape of haematological cancers, especially CLL [132], and can become a key driver of personalised cancer care [133].NGS is able to detect single-nucleotide variants (SNV), small structural changes, and balanced translocations, as well as to confirm CNV detected by array, by providing a base-to-base view of the genome [134] while GWAS is able to identify multiple low-risk variants that together explain about 16% of the familial risk of CLL other than detection of higher-risk SNPs or CNVs associated with disease risk in those families [135].NGS is also to detect gene mutation in TP53, ATM, NOTCH1, SF3B1, MYD88, and BIRC3; all the aforementioned genes are related to increased susceptibility of patients to develop CLL [58,136,137].The commonly used NGS platforms are Illumina HiSeq and Illumina MiSeq as well as Ion Torrent from Life Technologies.Data provided by array CGH and NGS technologies has significantly enhanced the knowledge of cancer biology and its underlying driver genes for pharmacogenetics and has guided targeted therapy development and drug-resistance prediction [61]. However, NGS and GWAS has its own pitfalls that need to be addressed.First, the massive amount of data that is obtained from the NGS and GWAS may not be relevant for a diagnostic setting.Second, high cost can be incurred from procurement of NGS equipment, software, and consumables.Third, NGS needs a specialised high-power computer and technician to analyse and store all the data obtained [62].Increased sensitivity is one of the main benefits of NGS methods for genetic diagnostics; however, so far, this method has only been applied to the detection of single-nucleotide variants (SNVs).Although some chromosomal fusions can be detected using NGS-based approaches with prior knowledge of translocation/fusion partners, a large portion of the genome is still unavailable for structural variant detection due to technical restrictions [138]. A major advantage of using whole-genome sequencing (WGS) is it can identify chromosome inversions and translocation.A study conducted by Robbe et al. (2022) using WGS identified 1248 inversions with frequent breakpoints involving either immunoglobulin light chain kappa (IGK), immunoglobulin heavy chain (IGH) locus, or ch13q14.2 and 993 translocations with no previously documented role in CLL, including t(14;22) with a breakpoint within WDHD1 and t(5;6) (CTNND2-ARHGAP18).Moreover, authors also identified STED2/del3p.21.31, del9p21.3, and gain of chr17q21.31are associated with relapsed/refractory (R/R) disease and TP53 disruption, whereas MED12 and DDX3X mutations are associated with unmutated IGH CLL [73].This technology has been reported successfully as not only capturing SNVs with a high level of accuracy but also working well for the detection of disease-causing CNVs.In addition, WGS has the capability of identifying chromosomal rearrangements, as well as STRs and ROH.It is interesting to note that the diagnosis rate of WGS in this study was 27%, which was much higher than the diagnostic rate of clinical microarray (12%) [139]. NGS and arrays are appropriate for cytogenomic studies across a variety of constitutional and cancer research applications, as NGS provides complementary detection capabilities.On a single piece of equipment referred to as the NextSeq 550 System, the researchers are able to carry out both NGS and array scanning.Genome visualisation is possible with the conventional molecular cytogenomic methods for evaluating chromosomal aberrations, such as FISH and karyotyping.However, these approaches often produce a low-resolution image of the genome.As a consequence, the results of such procedures are not always comprehensive [140].Cytogenomic microarrays provide not only a simple tool but also a reliable method for analysing chromosomal abnormalities at a higher resolution.High-quality microarrays from Illumina are available for the purpose of detecting chromosomal abnormalities while also providing precise and dependable cytogenomic data [140]. In Malaysia, there are a few centres that offers genetic testing in cancer, especially for haematological malignancies.Commonly, most centres will offer CCA and FISH for known chromosomal abnormalities in certain types of haematological malignancies as a tool for diagnosis.They also offer molecular testing (PCR) to detect common fusion genes that are involved in haematological malignancies, such as the BCR-ABL fusion gene in chronic myeloid leukaemia (CML), BCR-ABL fusion gene, TEL-AML1 fusion gene, and E2A-PBX1 fusion gene and MLL gene rearrangement in acute lymphocytic leukaemia (ALL), and PML-RARA gene in acute promyelocytic leukaemia.For array-based technologies such as DNA microarray and NGS, there are not many centres to choose from except for private companies.Furthermore, the array-based technologies are too costly (around ~MYR 2000-MYR 2500 per test) and the need for well-trained staff and experts to interpret the results make them not suitable to be the first-line diagnostic tool in haematological malignancies.However, as the Western countries and other Asian countries such as Korea, China, and Taiwan already used array-based technologies as first diagnostic tools, we need to improve our diagnostic tools so that we are in line with the current diagnosis developments, thus later contributing to better and more precise treatments [79]. This study is a pioneer in Malaysia for performing CLL profiling using a microarray platform using Affymetrix CytoScan 750K array chip; it hopefully will illustrate the genetic aberrations that are involved in CLL pathogenesis.The findings in this study are crucial, as many studies done previously by other populations have already acknowledged the difference of genomic aberrations between Asian CLL and Western CLL [80,141].Therefore, databases for CLL patients in Malaysia can be created based on these data. The current assay, called, the \"CytoTerra TM Platform\", elevates cytogenetics to new heights.This assay combines the genome-wide structural variation detection capability of conventional cytogenetics with the molecular-level precision of chromosomal microarrays (CMA) and FISH in a single, cost-effective manner with an NGS-based assay.The CytoTerra Platform uses ultra-long-range genome sequencing to assess the breadth of chromosome aberrations with greater resolution than conventional cytogenetic analysis, CMA, and FISH combined.The CytoTerra TM Platform possesses unique features such as genome-wide detection, the ability to detect complex rearrangements, the ability to identify unbalanced chromosomal alterations (deletion, duplication, and amplification), and the ability to examine balanced rearrangements (inversion, insertion, reciprocal, and Robertsonian translocation), and does not require specialised instrumentation [142,143].Table 4 highlights the advantages and disadvantages of each cytogenomics approach used to diagnose CLL. All genetic aberration data obtained from CCA, FISH, DNA microarray and wholegenome sequencing in CLL patients will help the clinician to tailor treatment according to patients' needs, reduce the complication of treatment, and improve survival outcomes [144,145].Moreover, according to [113], the most recent applications of cytogenomic techniques include conducting research on topologically associated domains, studying interchromosomal interactions, and chromoanagenesis, characterising the 3D structure of chromosomes in various tissue types and shedding light on the multilayer arrangement of chromosomes and the function of repetitive repeats and noncoding RNAs in the human genome. ", "section_name": "Cytogenomic Approaches in CLL: Advantages and Challenges", "section_num": "3." }, { "section_content": "The landscape of CLL genomics will become more thorough and precise with the help of technological evolution.Together with data collected from the DNA microarray technology as well as conventional cytogenetic, FISH, and other advanced technology, whole-genome sequencing may create a new pathway for creating potential therapeutic agents that are more focused on targeted therapy.Despite the fact that there were many methods to detect genomic aberration in CLL, microarray-based technology was deemed to be superior to others (CCA, FISH, MLPA, and PCR) and cost-effective compared to NGS and GWAS.Thus, laboratory technologists should be well-trained and well-versed with microarray technology to keep up with the latest technology.It also helps the clinicians to obtain more detailed data on the disease as well as to determine and quantify diseaseassociated genetic profiles and improve clinical diagnosis/prognosis, tumour classification, and ultimately, cancer therapy. ", "section_name": "Conclusions", "section_num": "4." } ]	[ { "section_content": "We would like to convey our appreciation to Universiti Sains Malaysia (USM) for funding our research (GPSP). ", "section_name": "Acknowledgments:", "section_num": null }, { "section_content": "Funding: This research was funded by the PPSP Incentive Postgraduate Studies Development Fund, School of Medical Sciences, USM (grant account number: 1001/PPSP/8070011).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable. ", "section_name": "", "section_num": "" }, { "section_content": "Data Availability Statement: Not applicable. ", "section_name": "", "section_num": "" }, { "section_content": "Author Contributions: S.S. contributed conceptualisation; W.N.W.M.Z.contributed to preparing the manuscript; N.M.Y., A.A.A.A., N.N.Z. and S.S. reviewed and edited the manuscript.All authors have read and agreed to the published version of the manuscript. The authors declare no conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null }, { "section_content": "Author Contributions: S.S. contributed conceptualisation; W.N.W.M.Z.contributed to preparing the manuscript; N.M.Y., A.A.A.A., N.N.Z. and S.S. reviewed and edited the manuscript.All authors have read and agreed to the published version of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare no conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.3324/haematol.10317	APRIL but not BLyS serum levels are increased in chronic lymphocytic leukemia: prognostic relevance of APRIL for survival	APRIL (a proliferation-inducing ligand) and BLyS (B lymphocyte stimulator) expression have been reported in chronic lymphocytic leukemia (CLL) cells. We examined APRIL and BLyS serum levels in CLL patients and evaluated the prognostic significance of APRIL expression on survival.	[ { "section_content": "BLyS is a fundamental survival factor for transitional and mature B cells, whereas APRIL mainly affects B-1 cell activity, humoral responses and immunoglobulin class switching. 1We recently reported that aging APRIL transgenic mice develop B-1 cell-associated tumors that are highly reminiscent of human CLL; our initial analysis showed increased levels of circulating APRIL in CLL patients (n=22) compared to healthy donors. 2 Two other reports described that BLyS and APRIL can act in an autocrine manner in CLL tumor cells, promoting cell survival. 3,4In addition, APRIL produced by inflammatory cells that infiltrate tumor lesions appears to contribute to disease in B cell lymphoma. 5o further evaluate APRIL and BLyS implication in CLL, we performed a retrospective study of a cohort of 95 patients diagnosed with CLL according to NCI criteria. 6fter informed consent was obtained, peripheral blood samples were collected from 1991 to 2005, aliquoted and frozen.As a control, 32 sera from age-and sex-matched healthy donors were processed in the same manner.The sera tested were of patients at different stages (A, B and C), but mostly (80%) of untreated patients at either stage A or B. Circulating APRIL was measured by ELISA as described 2 and the mean APRIL serum level was 10.5 ng/ml in healthy donors (range=1.8-25.3ng/mL; n=32) and 64.5 ng/mL in CLL patients (range=1.5-208.5 ng/mL; n=95; p<0.0001; Figure 1A); 75% of all CLL patients showed an increase in circulating APRIL protein compared to controls.We also observed significant differences in APRIL levels in CLL patients grouped by Binet's staging; the mean APRIL serum level was 53.5 ng/mL in stage A patients (n=57) and 79.9 ng/mL in B/C patients (n=37; p=0.02). By contrast, the CLL patient sera tested by ELISA (R&D Systems) showed a moderate decrease in circulating BLyS levels compared to healthy controls (Figure 1B, p<0.0001).This was similar to recent observations by Haiat et al. 7 Another study revealed increased BLyS serum levels only in CLL patients with a familial history of B-cell lymphoproliferative malignancies. 8In our study, information on patient familial history was not available.Whether the observed decrease of circulating BLyS levels is relevant for the development of CLL awaits further studies. To evaluate the clinical relevance of circulating APRIL on CLL, we divided the patient cohort according to the median APRIL serum level.Patients were considered APRIL high when serum APRIL concentration ≥56 ng/mL or APRIL low when APRIL concentration was <56 ng/mL.A description of our CLL patient population and distribution according to the median APRIL levels is shown in Table 1.Fisher's exact test showed no significant association between APRIL levels and Binet's stage or VH mutational status when all patients where considered.Nevertheless, when analyzing the association of APRIL levels and VH mutational status in subgroups defined by Binet's stage, we found that B/C patients with unmutated VH genes were associated with higher serum APRIL levels than B/C patients with VH mutated genes (p=0.04). We used Kaplan-Meier analysis to estimate the prognostic value of APRIL serum levels on overall survival (OS).APRIL high patients had a significantly poorer prognosis than those classified as APRIL low (survival probability of 53% and 94% respectively; p=0.003) (Figure 1C).Univariate Cox analysis confirmed that APRIL levels had a significant correlation with survival (p=0.02) and multivariate Cox analysis using the three variables; VH status, ", "section_name": "", "section_num": "" } ]	[ { "section_content": "+ These authors share senior authorship *Institut de Génétique Moléculaire de Montpellier, UMR5535, Montpellier, France; °Department of Statistics and Operation Research, University of Jaén, Spain; # Laboratory for Experimental Oncology and Radiobiology, Amsterdam Medical Center, Amsterdam, The Netherlands; @ Service d'Hématologie Biologique, Groupe Hospitalier La Pitié-Salpêtrière, Paris, France Funding: MH and LPC were supported by Action Concertee Incitative Jeunes Chercheurs (ACI), Association pour la Recherche sur le Cancer (ARC), Fondation Recherche Médicale (FRM), Fondation de France and Ramón y Cajal Program.JPM was sponsored by a Dutch Cancer Society grant (2003-2812).Acknowledgments: we thank Sylvie Baudet, Martine Brissard, Patrick Bonnemye and Myriam Boudjoghra for their invaluable help, and Catherine Mark for her editorial assistance.Key words: LLC, APRIL serum levels, overall survival, prognostic factor.Correspondence: Lourdes Planelles, PhD, Department of Immunology and Oncology, Centro Nacional de Biotecnologia/CSIC, Darwin 3, UAM Campus de Cantoblanco, 28049 Madrid, Spain.Phone: international +34.915854855.Fax: international +34.9.13720493.E-mail: [email protected] ", "section_name": "", "section_num": "" } ]
10.17816/kmj2020-145	Features of immunophenotypic finding B-cell lymphoproliferative diseases by flow cytometry	<jats:p>Aim. To assess the information content of conventional and additional immunophenotypic markers (CD200, CD305) in the differential diagnosis B-cell lymphoproliferative diseases by flow cytometry. Methods. An immunophenotypic study using 4-color flow cytometry was performed in 204 patients with different variants of B-cell non-Hodgkin's lymphomas. The study material included peripheral blood and bone marrow. The expression of CD45, CD19, CD20, CD22, CD79b, CD79a, CD5, CD10, CD23, FMC7, CD43, CD38, CD11c, CD103, CD25, CD 200, CD 305, light chains of immunoglobulins (kappa/lambda) using monoclonal antibodies (Becton Dickinson, USA) was evaluated. The intensity of antigen expression was assessed using mean fluorescence intensity (y. e.). Results. Conventional FMC7-positive expression revealed only half patients with different variants of leukemization of non-Hodgkin's lymphomas, whereas atypical positive expression of CD23 was observed in patients with marginal spleen lymphoma and follicular lymphoma in 27.3 and 28.6% of cases, respectively. In mantle cell lymphoma, expression of CD200 in B-cell was detected in a significantly smaller number of observations, accompanied by a significant decrease in the average intensity of CD200 fluorescence compared to B-cell chronic lymphocytic leukemia (B-CLL) cells. The mean fluorescence intensity (MFI) of CD305 in hairy cell leukemia is significantly higher than in splenic marginal zone lymphoma (SMZL) with villous lymphocytes. Conclusion. Different levels of the information content of some conventional markers were revealed in differential immunophenotypic diagnosis of B-cell lymphoproliferative diseases by flow cytometry; the use of additional markers CD200 and CD305 was highly informative in differential diagnostics between different variants of B-cell lymphoproliferative diseases with similar immunophenotypic and morphological characteristics of lymphoid elements.</jats:p>	[ { "section_content": "", "section_name": "Обмен клиническим опытом", "section_num": null }, { "section_content": "Methods.An immunophenotypic study using 4-color flow cytometry was performed in 204 patients with different variants of B-cell non-Hodgkin's lymphomas.The study material included peripheral blood and bone marrow.The expression of CD45, CD19, CD20, CD22, CD79b, CD79a, CD5, CD10, CD23, FMC7, CD43, CD38, CD11c, CD103, CD25, CD 200, CD 305, light chains of immunoglobulins (kappa/lambda) using monoclonal antibodies (Becton Dickinson, USA) was evaluated.The intensity of antigen expression was assessed using mean fluorescence intensity (y.e.).Results.Conventional FMC7-positive expression revealed only half patients with different variants of leukemization of non-Hodgkin's lymphomas, whereas atypical positive expression of CD23 was observed in patients with marginal spleen lymphoma and follicular lymphoma in 27.3 and 28.6% of cases, respectively.In mantle cell lymphoma, expression of CD200 in B-cell was detected in a significantly smaller number of observations, accompanied by a significant decrease in the average intensity of CD200 fluorescence compared to B-cell chronic lymphocytic leukemia (B-CLL) cells.The mean fluorescence intensity (MFI) of CD305 in hairy cell leukemia is significantly higher than in splenic marginal zone lymphoma (SMZL) with \"villous\" lymphocytes.Conclusion.Different levels of the information content of some conventional markers were revealed in differential immunophenotypic diagnosis of B-cell lymphoproliferative diseases by flow cytometry; the use of additional markers CD200 and CD305 was highly informative in differential diagnostics between different variants of B-cell lymphoproliferative diseases with similar immunophenotypic and morphological characteristics of lymphoid elements.Keywords: B-cell lymphoproliferative diseases, flow cytometry, СD200, СD305 markers.Преимущества метода многоцветной проточной цитометрии определяют широкие возможности его использования, прежде всего в диагностике и классификации лимфопролиферативных заболеваний (ЛПЗ) [1], мониторинге остаточного опухолевого клона, оценке факторов прогноза и резистентности к терапии.Традиционное морфологическое исследование при ЛПЗ не позволяет определить принадлежность опухолевых лимфоидных клеток к какой-либо линии (Т-, В-или NK) или стадии дифференцировки.Метод проточной цитометрии позволяет быстро выявить клональность опухолевых В-лимфоцитов, коэкспрессию основных для конкретной опухоли поверхностных и/или цитоплазматических маркёров.Однако не всегда иммунофенотипическая характеристика опухолевых лимфоцитов отдельных пациентов соответствует классической картине того или иного варианта В-ЛПЗ, что затрудняет интерпретацию полученных данных и может привести к неверному диагнозу. В некоторых публикациях [2][3][4][5] появились данные об использовании относительно новых моноклональных антител в диагностической панели для иммунофенотипирования В-ЛПЗ.Мы проанализировали информативность традиционных и дополнительных иммунофенотипических маркёров (CD200, CD305) при различных вариантах В-клеточных ЛПЗ.CD200 (OX2) -трансмембранный гликопротеид, относящийся к суперсемейству имму-ноглобулинов, экспрессируется на тимоцитах, покоящихся и активированных Т-лимфоцитах, В-лимфоцитах, дендритных, эндотелиальных клетках, нейронах, но отсутствует на NK-клетках, моноцитах, гранулоцитах, тромбоцитах.Опухолевые В-клетки характеризуются яркой гомогенной экспрессией CD200 при В-клеточном хроническом лимфолейкозе (ХЛЛ), волосатоклеточном лейкозе (ВКЛ), значительно слабее её выраженность при лимфоме клеток мантийной зоны (ЛКМЗ) [4,5].Экспрессию CD200 считают неблагоприятным прогностическим признаком при множественной миеломе [6][7][8]. CD305, или LAIR-1 (от англ.Leukocyte-Associated Ig-Like Receptor-1), -трансмембранный гликопротеид, относится к суперсемейству иммуноглобулинов, экспрессируется на значительной части Т-, В-, NK-лимфоцитов, моноцитах, дендритных клетках, тимоцитах.Экспрессию LAIR-1 отмечают на ранних стадиях дифференцировки В-лимфоцитов, но она отсутствует на плазмобластах и плазмоцитах.Кроме того, LAIR-1 может функционировать как ингибирующий рецептор на Т-и NK-лимфоцитах [9,10]. В рекомендациях Европейского консорциума по проточной цитометрии (EuroFlow) маркёр LAIR-1 включён в дифференциально-диагностическую панель В-ЛПЗ для диагностики ВКЛ (Leukemia, 2012). Цель исследования -оценить информативность традиционных и дополнительных имму-нофенотипических маркёров (CD200, CD305) при дифференциальной диагностике В-ЛПЗ методом проточной цитометрии. Иммунофенотипическое исследование проведено 204 пациентам с подозрением на В-ЛПЗ в научно-исследовательской лаборатории ГБУЗ МО «Московский областной научно-исследовательский клинический институт им.М.Ф.Владимирского».Также обследована группа из 20 пациентов с реактивным лимфоцитозом, у которых наличие В-ЛПЗ было исключено.Материалом для исследования служили периферическая кровь и костный мозг. Исследования были одобрены на заседании независимого комитета по этике ГБУЗ МО «Московский областной научно-исследовательский клинический институт им.М.Ф.Владимирского» (протокол №10 от 15.10.2015)и проведены с получением добровольного и информированного согласия пациентов. Возраст пациентов варьировал от 30 до 80 лет, средний возраст составил 56±15 лет. Иммунофенотипическое исследование проводили методом лазерной проточной цитометрии, применяя четырёхцветный проточный цитофлюориметр FACSCalibur (Becton Dickinson, США) с использованием программного обеспечения CellQwest, моноклональных антител, конъюгированных с флюоресцентными красителями (производство BD Biosciences, США).Использовали следующую панель моноклональных антител: Анти-CD45-FITC, Per-CP или Per-CP-Сy5,5, APC; анти-CD3-FITC, анти-CD19-APC, Per-CP или Per-CP-Сy5,5; анти-CD20-FITC, Per-CP или Per-CP-Сy5,5; анти-CD5, анти-FMC7, анти-CD38, анти-CD103, анти-CD43, анти-sIg kappa, меченные FITC; анти-CD(16+CD56), анти-CD10, анти-CD23, анти-CD56, анти-CD22, анти-CD79b, анти-CD25, анти-CD11c, анти-sIg lambda, меченные PE.Дополнительно в панель были включены моноклональные антитела анти-CD200 -PE (Clone MRC OX-104 BD Pharmingen), анти-CD305-PE (Clone DX26 BD Pharmingen). Иммунофенотипическое исследование проводили с применением стандартной методики пробоподготовки.Критерием позитивности считали наличие экспрессии антигена на поверхности или в цитоплазме более чем 20% опухолевых клеток.Оценку интенсивности экспрессии антигенов проводили по параметру средней интенсивности флюоресценции (MFI -от англ.Mean Fluorescence Intensity), выраженной в условных единицах (у.е.). Статистическая обработка полученных результатов проведена с использованием стати-стической программы Statistica 8.0 c расчётом средней величины и квадратичной ошибки средней (М±m).Для определения статистической значимости различий в средних значениях количественных показателей использовали t-критерий Стьюдента.Частоту антигенов в исследуемых когортах пациентов сравнивали по критерию Фишера. Из 204 обследованных пациентов ХЛЛ был диагностирован у 104, у 18 человек -ВКЛ, у 41 пациента -ЛКМЗ, у 34 больных -лимфома маргинальной зоны селезёнки (ЛМЗС), у 7 пациентов -лейкемизация фолликулярной лимфомы.Диагноз ЛКМЗ был подтверждён цитогенетическими исследованиями t(11;14) и/или выявлением экспрессии циклина D1 иммуногистохимическими методами. Данные по оценке традиционных иммунофенотипических маркёров при В-ЛПЗ представлены в табл. 1. Общий иммунофенотипический признак всех В-ЛПЗ -обнаружение рестрикции мембранных лёгких цепей иммуноглобулинов (kappa-либо lambda-тип), что служит подтверждением клональности В-лимфоцитов при опухолевой трансформации.Оценка интенсивности экспрессии CD20 по параметру MFI позволяет дифференцировать клетки В-ХЛЛ с низкой/слабой (dim) степенью экспрессии от остальных опухолевых клеток В-ЛПЗ, для которых характерна промежуточная (mod), либо высокая/яркая (bright) интенсивность экспрессии данной молекулы, что служит одним из основных традиционных дифференциальнодиагностических критериев, позволяющих отличить ХЛЛ от зрелоклеточных лимфом и ВКЛ.Общепринятым традиционным иммунофенотипическим признаком лейкемизации неходжкинских лимфом и ВКЛ считают позитивность FMC7, который обычно не экспрессирован при ХЛЛ, что также служит дифференциально-диагностическим критерием. Иммунофенотип лимфоцитов при ХЛЛ характеризовался классическими признаками: позитивной экспрессией антигенов CD5, CD23, CD43, отсутствием экспрессии CD10, FMC7.Все пациенты с ХЛЛ демонстрировали слабую (dim) экспрессию молекулы CD20, часть пациентов (4,5%) имели нетипичную яркую (bright) экспрессию CD22, у 33,8% пациентов обнаружено отсутствие мембранной экспрессии CD79b.У 14,4% больных не было выявлено рестрикции по мембранным лёгким цепям иммуноглобулинов.Отсутствие молекулы CD79b на поверхности клеток В-ХЛЛ может служить признаком делеции (13)(q14.3)или нарушения формиро-Обмен клиническим опытом вания В-клеточного рецепторного комплекса [11].26% пациентов демонстрировали позитивную экспрессию CD38 на клетках В-ХЛЛ, что расценивают как иммунофенотипический показатель неблагоприятного прогноза и резистентности к терапии [12].У 73% пациентов с ХЛЛ обнаружена позитивная экспрессия рецептора к интерлейкину-2 (CD25) на опухолевых В-лимфоцитах в диапазоне от 20 до 89% позитивных клеток.По данным литературы, у таких больных отмечают более агрессивное течение заболевания, есть связь с хромосомными аномалиями, стимуляцией Toll-like-рецепторов [13,14].По нашим данным, выраженная экспрессия CD25 на клетках В-ХЛЛ уже на этапе первичной иммунофенотипической диагностики служит маркёром высокой вероятности рефрактерности к проводимой терапии и низкого качества ремиссии заболевания, а также может быть показателем прогрессирования или развития рецидива заболевания [15]. Иммунофенотипический профиль опухоле вых В-лимфоцитов при лейкемизации ЛКМЗ характеризовался позитивной экспрессией CD5, отсутствием экспрессии CD23, CD43, CD10.У 20 и 15% пациентов с ЛКМЗ выявлена слабая (dim) экспрессия CD20 и CD22 соответственно, у 11,4% пациентов отсутствовала экспрессия CD79b.Позитивная экспрессия FMC7 выявлена только у 57,1% пациентов. Особой проблемой при иммунофенотипировании становится дифференциальная диагностика между CD5-позитивными ХЛЛ и ЛКМЗ при вариабельности антигенов CD23, реже CD43, а также при вариабельности интенсивности экспрессии CD20.Использование маркёра СD200 может помочь в разрешении этой проблемы.Оценка экспрессии СD200 у исследованных пациентов представлена в табл. 2 и 3. Во всех случаях ХЛЛ и в большинстве случаев реактивного лимфоцитоза выявлена позитивная экспрессия СD200 на В-лимфоцитах.Позитивная экспрессия СD200 обнаружена у пациентов с ЛКМЗ в значительно (p=0,000) меньшем количестве наблюдений, чем при ХЛЛ, что согласуется с данными литературы, хотя доля позитивных по СD200 пациентов с ЛКМЗ несколько выше, чем у других исследо- Для фенотипа опухолевых клеток фол ликулярной лимфомы была характерна яркая (bright) экспрессия всех пан В-клеточных маркёров в 100% наблюдений, позитивность CD10, вариабельность маркёров CD23, CD43 и CD38.В 43% случаев рестрикция лёгких цепей иммуноглобулинов выявлена только при внутрицитоплазматическом окрашивании.Позитивная экспрессия FMC7 обнаружена только у половины обследованных пациентов (57,1%), а позитивная экспрессия CD23 -в 28,6% наблюдений.В последней классификации Всемирной организации здравоохранения (2016) выделен новый вариант преимущественно диффузной CD23-позитивной фолликулярной лимфомы с фенотипом CD10 + bcl-2 + bcl-6 + , характеризующийся более агрессивным течением и del 1p36 [16]. Иммунофенотип В-лимфоцитов при ЛМЗС характеризовался преимущественно яркой (bright) экспрессией всех пан В-клеточных маркёров, отсутствием экспрессии CD5, CD10, CD43, CD103 и CD38, вариабельностью CD23, CD25 и FMC7.В частности, не характерная для данной группы пациентов позитивная экспрессия антигена CD23 обнаружена практически у трети (27,3%) больных, а позитивная экспрессия классического традиционного маркёра FMC7 выявлена только у половины (54,5%) пациентов. Опухолевые клетки при классической фор ме ВКЛ демонстрировали яркую (bright) экспрессию всех пан В-клеточных маркёров, отсутствие CD5, наличие позитивной экспрессии антигенов FMC7, CD11с, CD103 и CD25.Из 18 пациентов с иммунофенотипически верифицированным диагнозом ВКЛ у 27,7% нами установлена вариантная форма с позитивной Не было обнаружено достоверных различий по содержанию СD305-позитивных клеток между данными группами пациентов, но средняя интенсивность флюоресценции СD305 при ВКЛ была достоверно выше, чем при лейкемизации ЛМЗС (рис.3).Этот факт может быть использован при иммунофенотипической дифференциальной диагностике этих вариантов В-ЛПЗ со сходной морфологической характеристикой лимфоидных элементов.ВЫВОДЫ 1. Обнаружены некоторые особенности иммунофенотипической характеристики опухолевых клеток у пациентов с В-клеточными лимфопролиферативными заболеваниями. 2. От 7,5 до 20% пациентов с лейкемизацией лимфомы клеток мантийной зоны и лимфомы маргинальной зоны селезёнки могут демонстрировать нетипичную слабую (dim) интенсивность экспрессии CD20, CD22 и CD79b, что затрудняет дифференциальную диагностику с хроническим лимфолейкозом. 3. Позитивная экспрессия традиционного маркёра FMC7, характерная для лейкемизации зрелоклеточных лимфом и волосатоклеточного лейкоза, выявлена только у половины пациентов с лимфомой клеток мантийной зоны, фолликулярной лимфомой и лимфомой маргинальной зоны селезёнки. 4. Обнаружено относительно много наблюдений с нетипичной позитивной экспрессией CD23 у пациентов с фолликулярной лимфомой и лимфомой маргинальной зоны селезёнки (28,6 и 27,3% соответственно). 5. Позитивная экспрессия СD200 на опухолевых клетках выявлена у пациентов с лимфомой клеток мантийной зоны в значительно (p <0,0001) меньшем количестве наблюдений, чем при хроническом лимфолейкозе и других вариантах В-клеточных лимфопролиферативных заболеваний. 6. Cтатистически значимое (p <0,001) снижение средней интенсивности флюоресценции СD200 при лимфоме клеток мантийной зоны по сравнению с хроническим лимфолейкозом даёт возможность использования данного критерия в дифференциальной диагностике этих вариантов В-клеточных лимфопролиферативных заболеваний у больных с высоким содержанием СD200-позитивных опухолевых В-клеток. 7. Показатель средней интенсивности флюоресценции СD305 при волосатоклеточном лейкозе достоверно выше, чем при лейкемизации лимфомы маргинальной зоны селезёнки, что может быть использовано при дифференциальной диагностике этих вариантов В-клеточных лимфопролиферативных заболеваний со сходной морфологической характеристикой лимфоидных элементов. Авторы заявляют об отсутствии конфликта интересов по представленной статье. ЛИТЕРАТУРА ", "section_name": "CD305) in the differential diagnosis B-cell lymphoproliferative diseases by flow cytometry.", "section_num": null } ]	[]
10.1371/journal.pone.0119723	Epidermal Growth Factor Receptor Inhibition Reduces Angiogenesis via Hypoxia-Inducible Factor-1α and Notch1 in Head Neck Squamous Cell Carcinoma	Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC). The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR) inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α) was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO) mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.	[ { "section_content": "Head and neck squamous cell carcinoma (HNSCC) ranks as the sixth most frequent cancer worldwide with approximately 500,000 new cases per year worldwide [1].Previous studies have established that risk factors, such as alcohol drinking, smoking, and human papilloma virus infection, contribute to the development of this fatal disease [2].However, the five-year survival rate of HNSCC patients remains relatively unchanged at 40% to 50% during the past three decades [3].Advanced-stage HNSCC patients have poor prognosis and often need both chemotherapy and radiotherapy [4].However, only 30% of advanced-stage HNSCC patients survive for more than 5 years.Important factors that contribute to this scenario include the relative hypoxic and angiogenic conditions of high tumor burden in HNSCC.These conditions promote the stemness of cancer stem cells with both local and distant metastatic potentials [5]. Emerging basic, preclinical, and clinical findings indicated that epidermal growth factor receptor (EGFR)-mediated aberrant signaling transduction is crucial in HNSCC tumorigenesis and progression [6].EGFR has been observed in 70% to 100% of all HNSCC lesions [7].The high phosphorylation status of EGFR is frequently correlated with poor prognosis [8].Activated EGF/EGFR pathway may promote cell proliferation, differentiation, angiogenesis, and antiapoptosis in HNSCC tumorigenesis and progression through the phosphoinositide-3-kinase (PI3K)/Akt, ras/raf/extracellular regulated protein (Erk), and signal transducer and activator of transcription pathways [9,10].Cetuximab is a chimeric IgG1 monoclonal antibody that is currently licensed for the treatment of HNSCC patients [11,12].This drug is used alone or in combination with chemotherapy as the first and second lines of treatment for advanced-stages patients [13].Hypoxia-inducible factor-1α (HIF-1α) is a principal molecular mediator for tumor angiogenesis, and Notch pathway dysregulation is a leading genetic instability in HNSCC [14][15][16].Previous reports suggested that the interaction between HIF-1α and Notch1 can influence tumor angiogenesis [17].However, the mechanism by which the interaction between EGFR and HIF-1α or Notch1 in HNSCC regulates angiogenesis and tumorigenesis has yet to be elucidated. In our previous studies, we established that Tgfbr1 and Pten conditional knock out (2cKO) mice demonstrate spontaneous fast HNSCC tumorigenesis with 100% penetration [18].HNSCC mice are highly angiogenic as compared with Pten knock out HNSCC mice [19].The present study shows that the overexpression and high phosphorylation of EGFR are crucial for the tumorigenesis of transgenic mouse models with combined Tgfbr1 and Pten loss.Furthermore, the cetuximab-induced inhibition of EGFR repressed tumor burden in xenograft HNSCC models.Chemopreventive treatment with cetuximab delays HNSCC onset in Tgfbr1/ Pten 2cKO mice and reduced HIF-1α-and Notch1-mediated angiogenesis.EGFR overexpression was correlated with HIF-1α and micro vessel density (MVD) in HNSCC clinical specimens.Thus, HIF-1α-and Notch1-mediated angiogenesis may be important for EGFR activation and may partially contribute to EGFR inhibitor sensitivity. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "All chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless indicated.Antibodies against EGFR, p-EGFR Tyr1068 , HIF-1α, and Notch1, Notch1 intracellular domain (NICD), Hes1, VEGF, Histone H3 were obtained from Cell Signaling Technologies (Danvers, MA, USA), CD31 were obtained from BD Pharmingen (NJ, USA).Cetuximab was purchased from Merck (Darmstadt, Germany).N-[N-(3,5-difluorophenacetyl-l-alanyl)]-Sphenylglycine t-butyl ester (DAPT, γ-secretase inhibitor which inhibited cleavage of Notch1) was obtained from Sigma-Aldrich (St. Louis, MO, USA). ", "section_name": "Chemicals and reagents", "section_num": null }, { "section_content": "The CAL27 cell line was purchased from ATCC and cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% FBS as previous described [20], in a humidified atmosphere of 95% air, 5% CO 2 at 37°C.CAL27 cells were serum-deprived for 12h and then treated with or without cetuximab (10 μg/ml) or DAPT (20 μM) in for indicated time (12h) in Anoxomat chambers (Mart Microbiology, Lichtenvoorde, the Netherlands) with appropriate oxygen concentrations for hypoxia (1% O 2 ) or normoxia (21% O 2 ).The cells were washed by phosphate buffer solution (PBS) two times and continue grow in serum-deprived endothelial basic medium (EBM, Lonza, Walkersville, MD, USA) medium for another 24h, and the cleared supernatants were collected as conditional medium (CM) and stored at -80°C.Pooled human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and cultured as previous described [19].In vitro wound healing assay and Boyden chamber transwell migration assay and tube formation assay of HUVECs were performed as previous described [19] with detail in Supplementary Material and Methods in S1 File. ", "section_name": "Cell culture, conditional medium collection and in vitro migration assay", "section_num": null }, { "section_content": "RNA interference were performed as previous described [20].Briefly, CAL 27 cells were seeded in 6cm culture dishes and allowed to grown to 80% confluence, transfected with TGFBR1 siRNA or/and PTEN siRNA with Hiperfect transfection reagent (Qiagen) according to the manufacturer's instruction.The knock down efficiency with at least 84% decrease of TGFBR1 or PTEN protein at a indicated time (24h) were confirmed by western blot as previous described [20].The expression of EGFR, p-EGFR Tyr1068 after the transfection was confirmed by Western blots. ", "section_name": "RNA interference", "section_num": null }, { "section_content": "Immunofluorescence were performed as previous described [19] and detail described in Supplementary Material and Methods in S1 File.Cells immunofluorescence was photographed by microscopy (CLSM-310, Zeiss, Germany). ", "section_name": "Cell immunofluorescence and confocal microscopy", "section_num": null }, { "section_content": "The nuclear/cytosolic fractionation of CAL27 cells was extracted using a Nuclear-Cytosol Extraction Kit (Applygen Technologies, Beijing, China) according to the manufacturer's instructions.Briefly, CAL27 cells treated with or without cetuximab were collected by centrifugation and resuspended in cytosol extraction buffer A. After incubation on ice for 10 min, the cells were mixed with cytosol extraction buffer B and further incubated on ice for 1 min.The lysates were separated by centrifugation, and the supernatant (cytosol extract) was collected and transferred into a new tube.The pellet was washed with cytosol extraction buffer A, and resuspended in cold nuclear extraction buffer.After incubation at 4°C for 30 min with constant rotation, the suspension was centrifuged at 12,000 g at 4°C for 5 min to collect the nuclear extract in the supernatant fraction.The nuclear and cytoplasmic extracts were subjected to Western blots analysis. ", "section_name": "Nuclear/cytosolic fractionation", "section_num": null }, { "section_content": "All animal studies include nude mice and transgenic mice were approved and supervised by Animal Care and Use Committee of Wuhan University and conducted in accordance with the NIH guidelines for the Care and Use of Laboratory Animals.Female athymic nude mice (18-20 g; 6-8weeks of age) were obtained from the Experimental Animal Center of Wuhan University in pressurized ventilated cage according to institutional regulations.Mice were housed in appropriate sterile filter-capped cages and with an inverse 12 h day-12 h night cycle.Lights were turned on at 8:30 am at 22 ± 1°C and 55 ± 5% humidity in the Experimental Animal Center of Wuhan University.All cages contained wood shavings, bedding and a cardboard tube for environmental enrichment.Animals fed and watered ad libitum. For heterotopic xenograft, nude mice were injected subcutaneously with CAL27 cells (4×10 6 in 0.2 ml of serum-free medium) in the flank when cells exponentially grow.After tumors were established, the mice were divided into two groups randomly, which were received cetuximab (10 mg/kg i.p. twice per week; n = 5) or normal saline (vehicle, 100ul i.p. 2/week; n = 5) infusion for 3 weeks.Tumor growth was determined by measuring the size of the tumors 3 times per week.The formula (width 2 ×length)/2 was used to determine tumor volumes.All mice were monitored daily for abnormal behavior, e.g., inability to eat or drink, unable to run away when touched, no response to stimuli.There was no mice which was euthanized before the experimental endpoint.The maximum tumour sizes reached to 1.2 cm during the course of this assay.The mice were euthanized using CO 2 and the tumors were harvested for the following immunohistochemical analysis and western blots analysis. ", "section_name": "Establishment and cetuximab treatment of CAL27 heterotopic xenograft tumors model in nude mice", "section_num": null }, { "section_content": "The squamous epithelial tissue specific and time inducible combined Tgfbr1/Pten knockout mice (Tgfbr1/Pten 2cKO, K14-Cre ERtam ; Tgfbr1 flox/flox ; Pten flox/flox ) were maintained as previously described [18,21].The Tgfbr1/Pten 2cKO mice and their vehicles (Tgfbr1 flox/flox ; Pten flox/flox ) were from the same litter with mixed genetic background of C57BL/6; FVBN; CD1;129.Five day consequent tamoxifen oral gavage need to applied to knock out Tgfbr1/Pten in oral epithelial and head neck skin.The tamoxifen application procedure has been previously described [18,21].Only 4-to 8-week-old male and female Tgfbr1/Pten 2cKO mice were included in this study.For in chemopreventive assay, 2 weeks after the last dose of oral tamoxifen application of the Tgfbr1/Pten 2cKO mice were randomized into a vehicle group (100ul PBS.i.p. n = 5 mice) or a cetuximab group (10 mg/kg i.p. twice per week, n = 6 mice), based on our pilot study on the tumorigenesis and survival of 2cKO mice.All mice were monitored daily for abnormal behavior, e.g., inability to eat or drink, unable to run away when touched, no response to stimuli.There was no mice which was euthanized before the experimental endpoint.The maximum tumour sizes reached to 1.0 cm during the course of this assay.At the end of studies, mice were euthanized using CO 2 , tissues were harvest for histology immunohistochemical analysis and western blots analysis.. ", "section_name": "Chemopreventive study on Tgfbr1/Pten combined conditional knockout (2cKO) mice", "section_num": null }, { "section_content": "For mouse phospho-RTK detection, we collected tissue of Tgfbr1/Pten 2cKO mouse tongue (n = 5), Tgfbr1/Pten 2cKO mouse tongue squamous cell carcinoma (n = 5), and their vehicles (Tgfbr1 flox/flox /Pten flox/flox tongue; n = 5) 6 weeks after the last oral tamoxifen dose.Antibody array was purchased from R&D system (proteome profiler mouse phospho-RTK array kit, ARY014).This array can detect the relative phosphorylation of 39 RTKs.Briefly, bovine serum albumin blocked the membrane containing immobilized phospho-RTK on a rocking platform at room temperature for 1 h.The membrane was then incubated with lysates of Tgfbr1/Pten 2cKO mouse tongue (n = 5), Tgfbr1/Pten 2cKO mouse tongue squamous cell carcinoma (n = 5), and their vehicles (Tgfbr1 flox/flox /Pten flox/flox tongue; n = 5) with Detection Antibody Cocktail overnight at 2°C to 8°C on a rocking platform.The membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Pierce Chemical, Rockford, IL) and then with chemiluminescent detection reagent.The membrane was scanned, and pixel density was presented by quantifying the mean spot densities from two experiments.For western blot, we collected tissue of Tgfbr1/Pten 2cKO mouse tongue (n = 2), Tgfbr1/Pten 2cKO mouse tongue squamous cell carcinoma (n = 5), and their vehicles (Tgfbr1 flox/flox /Pten flox/flox tongue; n = 2). ", "section_name": "Mouse phospho-Receptor Tyrosine Kinase (RTK) detection", "section_num": null }, { "section_content": "HN803 tissue arrays which contain 10 cases of normal tongue mucosa, 4 cases of lymph node metastasis and 57 confirmed cases of HNSCC were obtained from Biomax US (Rockville, MD, USA).The tissue array clinical data, including pathological classification and TNM classification were also provided by Biomax. ", "section_name": "Human HNSCC tissues array", "section_num": null }, { "section_content": "Antibodies against EGFR (1:50), p-EGFR Tyr1068 (1:200), HIF-1α, and Notch1, Hes1 (1:400) were stained in sections of xenograft samples and EGFR (1:50), HIF-1α, and Hes1 (1:400) were stained in sections of Tgfbr1/Pten 2cKO tongue SCC samples by immunohistochemistry.The methods and processes were described as previously reported [20].CD31 were stained in both xenograft and Tgfbr1/Pten 2cKO tongue SCC samples by frozen section immunohistochemistry.All slices were scanned using an Aperio ScanScope CS scanner with background substrate for each slice, and quantified using Aperio Quantification software (Version 9.1) for membrane, nuclear, or pixel quantification.Four random areas of interest were selected either in the epithelial or the cancerous area for scanning and quantification.Histoscore of membrane and nuclear staining was calculated as a percentage of different positive cells using the formula (3+)×3+(2+)×2+(1+)×1. Histoscore of pixel quantification was calculated as total intensity/ total cell number.The threshold for scanning of different positive cells was set according to the standard vehicles provided by Aperio. ", "section_name": "Histology, immunohistochemistry and scoring system", "section_num": null }, { "section_content": "Western blot were performed as previously described [22] with detail in Supplementary Material and Methods in S1 File. ", "section_name": "Western blot analysis", "section_num": null }, { "section_content": "Graph Pad Prism version 5.00 for Windows (Graph-Pad Software Inc) was used for data analyses.Student t tests were performed to analyze the differences between two groups.Two-way ANOVA analysis was used for analyzing differences between animal treatment results.Twotailed Pearson statistics were performed to correlate expression of EGFR with CD31, HIF-1α after confirmation of the sample with Gaussian distribution.All value was exhibited as Mean values ± SEM.P<0.05 were considered statistically significant. ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Tyrosine kinase dysregulation, overexpression and high activation are common phenomena in different cancers, including HNSCC.To examine the possible tyrosine kinase overexpression in the Tgfbr1/Pten 2cKO mouse model of HNSCC, we used a high-throughput antibody array with 39 RTKs to test the RTK expression of Tgfbr1/Pten 2cKO mouse tongue SCC in comparison with those of Tgfbr1/Pten 2cKO mouse tongue and Tgfbr1 flox/flox /Pten flox/flox tongue.Results revealed that the tyrosine kinases of EGFR, ErbB2, macrophage-stimulating protein receptor (MSPR) and platelet-derived growth factor receptor alpha (PDGFα) were highly expressed in Tgfbr1/Pten 2cKO mouse tongue SCC (Fig. 1A and1B).Particularly, EGFR overexpression seemed to be the predominant molecular event in mouse tongue SCC (Fig. 1A and1B).To confirm antibody array results, we used immunohistochemistry to directly observe the expression of EGFR in Tgfbr1/Pten 2cKO mouse tongue SCC.As shown in Fig. 1C, EGFR was almost negative in Tgfbr1 flox/flox /Pten flox/flox mucosa.The staining of EGFR in Tgfbr1/Pten 2cKO mouse HNSCC was even evidently stronger than that in in Tgfbr1/Pten 2cKO mouse mucosa (Fig. 1C).The results from western blots analysis (Fig. 1D) also validated this finding.More importantly, the activation of EGFR, p-EGFR Tyr1068 was much higher in Tgfbr1/Pten 2cKO mouse tongue SCC than that in the vehicle.Given that the mouse model was generated by conditionally knocking out Tgfbr1/Pten, we hypothesized that the expression levels of either EGFR or p-EGFR Tyr1068 increased after the knock down of TGFBR1 and PTEN, or both of them in vitro.The expression and activation of EGFR increased when the tongue cancer cells CAL27 were transfected with TGFBR1 and/or PTEN in siRNA (Fig. 1E).These results strongly indicate that tyrosine kinase dysregulation, particularly EGFR, is an important molecular event in the Tgfbr1/Pten 2cKO mouse model of HNSCC carcinogenesis and the deletion of Tgfbr1 or Pten increased the expression and phosphorylation of total EGFR. ", "section_name": "High EGFR expression in the Tgfbr1/Pten 2cKO mouse model of HNSCC", "section_num": null }, { "section_content": "We treated heterotopic xenograft tumors derived from CAL27 cells with cetuximab to further identify the possible function of EGFR in HNSCC development.The mice received the treatment at 21 d post implantation and were euthanized for Western blot and immunohistochemical analyses on day 42.Cetuximab significantly delayed tumor growth (Fig. 2A and2B).Fig. 2C showed the growth curves in tumors treated with cetuximab or vehicle, The mice administered with cetuximab showed partial tumor regression after 8 d of treatment.,The cetuximab-treated mice showed significant tumor inhibition after 12 d of treatment (P < 0.01) compared with the vehicle-treated group.We harvested and weighted the tumor at end point of experiment and results revealed cetuximab possessed antitumor activity because the tumor in the vehicle-treated group had significantly higher weight than those in the cetuximabtreated group (Fig. 2D).The indicated dose of cetuximab exerted no significant toxicity to the mice because the mice weight between cetuximab-and vehicle-treated groups showed no significant difference (Fig. 2E).These results demonstrated that EGFR blockade effectively prevented tumor growth. ", "section_name": "Cetuximab treatment of CAL27 heterotopic xenograft tumors", "section_num": null }, { "section_content": "We performed a chemopreventive study on Tgfbr1/Pten 2cKO mice to determine whether or not an increase in EGFR was an early event in HNSCC tumorigenesis.We induced the onset of HNSCC tumor in Tgfbr1/Pten 2cKO mice as previously described [20].The induction and drug administration strategies were shown Fig. 3A.Two weeks after the last tamoxifen oral gavages, the mice were treated with EGFR inhibitor or vehicle for 2 weeks.Cetuximab significantly (P < 0.001, n = 6) delayed tumorigenesis in external head and neck (Fig. 3B with quantification in Fig. 3D) and oral tongue tumors (Fig. 3C) in Tgfbr1/Pten 2cKO mice as compared with the vehicle group (n = 5).No significant weight loss was observed, indicating that cetuximab exerted no significant toxicity to these immuno-sufficient mice (Fig. 3E).These data indicated that EGFR blockade by cetuximab delayed the onset of HNSCC in 2cKO mice. ", "section_name": "Targeting EGFR by cetuximab delays HNSCC onset in Tgfbr1/Pten 2cKO mice", "section_num": null }, { "section_content": "Digital pathology was performed to explore whether or not EGFR inhibition influences angiogenesis in 2cKO mice.Immunohistochemical staining showed that cetuximab downregulated EGFR, p-EGFR, and MVD in the xenograft tissues of CAL27 cells.Quantification of histoscore by using Aperio digital pathology validated the observation results.(S1A and S1B Fig. ).The inhibition of EGFR expression and phosphorylation was also confirmed by Western blot (S1C Fig. ).We collected the conditioned medium (CM) after pretreating CAL27 cells with cetuximab.We performed an in vitro migration assay to further confirm the function of cetuximab in angiogenesis in vitro.As shown in Fig. 4A, the CM from cetuximab-pretreated CAL27 cells reduced HUVEC migration as compared with the vehicle medium.Similar results were obtained in the Boyden transwell migration assay and tube formation assay under both hypoxic and normoxic culture conditions (Fig. 4B and4C).The findings exhibited that CM significantly decreased HUVEC migration and tube formation after cetuximab pretreatment under both normoxic and hypoxic conditions when compared with the negative vehicle (Fig. 4D).Hypoxic culture conditions increased HUVEC migration as compared with normoxic culture conditions.The protein expression of HIF-1α and VEGFA were validated by western blots.Cetuximab reduced HIF-1α expression in normoxia and down-regulated VEGFA even in hypoxic condition (Fig. 4E).We further confirmed that 24 h of treatment with 10 μg/ml cetuximab reduced HIF-1α nuclear translocation in CAL27 cells under hypoxic culture conditions (Fig. 4F).To further detect the expression of HIF-1α, the protein levels expression of HIF-1α in the cytoplasmic and nuclear extracts were examined.As shown in Fig. 4G, cetuximab reduced expression HIF-1α in the nucleus in a concentration-dependent manner as compared with those in cells treated with vehicle.Aligned with this observation, cetuximab significantly inhibited HNSCC angiogenesis, and reduced HIF-1α nuclear translocation may be involved in this phenomenon. ", "section_name": "Cetuximab inhibits tumor-induced angiogenesis in vitro and in vivo", "section_num": null }, { "section_content": "To further confirm whether Notch1 signaling pathway was involved in the preventive effect of cetuximab on tumor-induced angiogenesis, endothelial function assays were performed in the presence of DAPT, a widely used inhibitor for Notch1.As shown in Fig. 5A to 5C, the CM from cetuximab-or DAPT-pretreated CAL27 cells reduced HUVEC migration as compared with the vehicle medium using wound healing assays.HUVECs migration even were further inhibited when treated with the CM from Cetuximab combined with DAPT.Similar results were obtained in the Boyden transwell migration assays and tube formation assays (Fig. 5A to 5C).We next detected the expression of NICD, a cleaved fragment that transduced activated signals of Notch1, and VEGFA by western blots (Fig. 5D).The results showed that the DAPT or cetuximab reduced the expression of NICD as well as VEGFA.More, cetuximab further reduced the expression of VEGFA even in the presence of DAPT, may suggesting other downstream molecule moderated VEGFA either.To explore the interaction between HIF-1α and Notch1, protein levels of HIF-1α and NICD were tested by western blots.And we found hypoxia up-regulated the activation of Notch1 consistent with the up-regulation of HIF-1α, while DAPT showed no effect on HIF-1α in hypoxia (Fig. 5F), suggesting HIF-1α might play as upstream of Notch1 at least in CAL27 cell lines. ", "section_name": "Notch1 signaling pathway is involved in cetuximab-reduced angiogenesis in vitro", "section_num": null }, { "section_content": "We next evaluated the immunoreactivity of EGFR to HIF-1α and CD31 in human tissue array to further assess the correlation of EGFR with HIF-1α and MVD in human HNSCC.Of 54 cases, 48 presented positive membrane staining in almost all epithelial tumor areas of HNSCC tissue; only 10% of the mucosa core showed staining, and this staining was limited in the basal layer (Fig. 6A).Hypoxia is a common phenomenon in HNSCC.Intense HIF-1α nuclear staining was observed in a large proportion of tumor cells, suggesting hypoxia is a common phenomenon in HNSCC.The.staining of HIF-1α was considerably strong in invasive cancer.Most human HNSCC lesions were also highly angiogenic, as reflected by the strong staining of the vascular endothelial marker CD31 (Fig. 6A).EGFR expression positively correlated with high expression levels of HIF-1α (P = 0.0001, r = 0.4192) and CD31 (P < 0.0001, r = 0.4296) (Fig. 6B; statistic including normal mucosa and HNSCC, n = 71).These results further confirmed that increased EGFR expression was significantly associated with hypoxia and angiogenesis in HNSCC ", "section_name": "Increased EGFR expression is related to HIF-1α and MVD in human HNSCC tissue", "section_num": null }, { "section_content": "We also examined the correlation of EGFR with Notch, another putative angiogenic molecule.Immunohistochemical staining showed that cetuximab treatment significantly reduced HIF-1α, Notch1, and Hes1 (putative downstream target of Notch1) (S2A and S2B Fig. Similar results were observed in 2cKO mouse HNSCC tissues, which are angiogenic and mimic human HNSCC in histological and molecule-expression patterns.Compared with the vehicle group (n = 7 from 5 mice), the residual cetuximab-treated HNSCC (n = 9 from 6 mice) showed downregulated HIF-1α, Hes1, EGFR, and CD31 expression (P < 0.001, Fig. 7A with quantification in Fig. 7B).The inhibition of EGFR expression and activation, HIF-1α, Hes1, VEGFA were also confirmed by western blots (Fig. 7C).These data further demonstrated that cetuximab downregulated tumor-induced angiogenesis in the 2cKO mouse model of HNSCC by inhibiting the HIF-1α and Notch1 pathways. ", "section_name": "Cetuximab inhibits tumor-induced angiogenesis by downregulating HIF-1α and Notch1", "section_num": null }, { "section_content": "Understanding the molecular mechanisms underlying HNSCC initiation and tumor evolution is important to delay tumor progression.Among the signaling events in HNSCC, the persistent overexpression and activation of EGFR have emerged as putative drug targets for HNSCC treatment in preclinical and clinical investigations [23][24][25].EGFR inhibitors, including cetuximab and lapatinab, can dramatically reduce tumor burden in HNSCC animal models [26] or patients [11] In the present study, the EGFR pathway is frequently activated in Tgfbr1/Pten 2cKO mice.EGFR overexpression may be related with Tgfbr1 and Pten downregulation.We assessed EGFR inhibition and angiogenesis in xenograft and transgenic mouse models of HNSCC.Results showed that EGFR inhibition with cetuximab can reduce tumor growth and angiogenesis in HNSCC. Stroma and immune cells serve important functions in tumor angiogenesis [27].Thus, the implantation of human HNSCC cells in immunodeficient mice may not completely reflect the clinical situation and may not accurately evaluate the efficacy of the drug on HNSCC angiogenesis [28].Tgfbr1/Pten 2cKO mice are characterized by 100% penetrance; in addition, they mimic human HNSCC with similar morphology and molecular alteration.Therefore, we analyzed the effect of EGFR on angiogenesis using this mouse model.Results showed that EGFR inhibitors at clinically relevant doses can reduce the regulation of HIF-1α and Notch1 in this tumor type with limited side effects.This phenomenon resulted in reduced angiogenesis and tumor shrinkage. In previous studies, we proved that the angiogenesis in 2cKO mouse HNSCC is related to HIF-1α activation by miR-135b [19].Herein, the blockade of EGFR in this experiment rapidly decreased HIF-1α, a hypoxic biomarker frequently observed in advanced-stage HNSCC [29].This effect likely involves the impact of cetuximab on angiogenesis by reducing HIF-1α nuclear translocation and/or reducing migration and chemoattractants, such as vascular endothelial growth factor A (VEGFA), for endothelial cells.This phenomenon prevents angiogenic signaling.The Notch signaling pathway is involved in the regulation of stem cell and neuronal cell death [30,31].However, recent evidence has shown that the Notch signaling pathway serves an important function during blood vessel formation and remodeling [32].The Notch signaling pathway is involved in endothelial cell biology; it influences the budding of endothelial tip cells during angiogenesis initiation [33].Notch1 was confirmed to be regulated by HIF-1α in a culture cell system [34].Notch blockade can abolish the tumor resistance of glioblastoma to VEGF inhibitors [35,36].Blocking both Dll4/Notch and VEGF pathways synergistically inhibits tumor growth, which indicates the potential application of Notch inhibitors as new adjuvant chemotherapy reagents [37].Dll4/Notch transcription was activated by Erk and PI3K signaling pathways, which were also downstream of canonical EGFR transduction [38].Notch1 downregulation also reduced VEGF expression [39].Thus, we hypothesized that cetuximab can decrease VEGF production and reduce HNSCC tumor angiogenesis by inhibiting the Notch signaling pathway.The present results showed that cetuximab inhibited the Notch1 signaling pathway by decreasing Notch1, Hes1, and VEGF expression in both nude mouse xenograft and 2cKO mouse models.Although these possibilities remain to be proven, the present findings support a unique anti-angiogenic function of cetuximab.That is, cetuximab can exert its antitumor activity by decreasing primary tumor growth and size, reducing HIF-1α instability, preventing endothelial cell initiation and migration, and downregulating VEGFA.These phenomena lead to the prevention of HNSCC angiogenesis. High HIF-1α expression in HNSCC tissue is an important factor that predicts poor prognosis and resistance to chemotherapy and/or radiotherapy.The clinical application of EGFR as a molecular target of HNSCC therapy is a revolutionary event.However, the radiosensitization mechanism of cetuximab, a new adjuvant chemo-radiotherapy of HNSCC, still warrants further investigation.The emerging preclinical and clinical information about the promising beneficial angiogenetic effects of cetuximab on HNSCCs and our present findings on the capacity of cetuximab to downregulate Notch1 and HIF-1α signaling benefit HNSCC therapy.We can envision that the present study and prior reports may provide a rationale for the future clinical evaluation of cetuximab in an adjuvant setting, as a part of a molecular-targeted strategy after definitive treatment. ", "section_name": "Discussion", "section_num": null } ]	[ { "section_content": "We thank Dr. Ashok B.Kulkarni of LCDB, National Institute of Dental and Craniofacial Research, NIH, USA for gift Tgfbr1/Pten 2cKO mice.We thank EssayStar for its linguistic assistance during the preparation of this manuscript. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "This work was funded by National Natural Science Foundation of China (81072203, 81272963) to Z.-J.Sun, (81371106) to L. Zhang, (81272946) to W.-F. Zhang, and (81170977) to Y.-F.Zhao.Z.-J.Sun is supported by program for new century excellent talents in university (NCET-13-0439), ministry of education of China.Support also came from National Natural Science Foundation of China (81472528) to Z.-J.Sun.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "All relevant data are within the paper and its Supporting Information files. ", "section_name": "", "section_num": "" }, { "section_content": "", "section_name": "Supporting Information", "section_num": null } ]
10.1515/jomb-2015-0017	Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia	<jats:title>Summary</jats:title> <jats:p><jats:bold>Background:</jats:bold> In chronic lymphocytic leukemia (CLL), <jats:italic>in vivo</jats:italic> apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the <jats:italic>Bcl2</jats:italic> family members play a central role.</jats:p> <jats:p><jats:bold>Aim:</jats:bold> The aim of this study was to investigate the association of pro-apoptotic <jats:italic>Bax</jats:italic> gene expression and <jats:italic>Bcl2/Bax</jats:italic> ratio with the clinical features of CLL patients as well as with molecular prognostic markers, namely the mutational status of rearranged immunoglobulin heavy variable (IGHV) genes and lipoprotein lipase (<jats:italic>LPL</jats:italic>) gene expression.</jats:p> <jats:p><jats:bold>Methods:</jats:bold> We analyzed the expression of <jats:italic>Bax</jats:italic> mRNA and <jats:italic>Bcl2/Bax</jats:italic> mRNA ratio in the peripheral blood mononuclear cells of 58 unselected CLL patients and 10 healthy controls by the quantitative reverse-transcriptase polymerase chain reaction.</jats:p> <jats:p><jats:bold>Results:</jats:bold> We detected significant <jats:italic>Bax</jats:italic> gene overexpression in CLL samples compared to non-leukemic samples (p=0.003), as well as an elevated <jats:italic>Bcl2/Bax</jats:italic> ratio (p=<0.001). Regarding the association with prognostic markers, the <jats:italic>Bcl2/Bax</jats:italic> ratio showed a negative correlation to lymphocyte doubling time (r=−0.307; p=0.0451), while high-level <jats:italic>Bax</jats:italic> expression was associated with <jats:italic>LPL</jats:italic>-positive status (p=0.035). Both the expression of <jats:italic>Bax</jats:italic> and <jats:italic>Bcl2/Bax</jats:italic> ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance.</jats:p> <jats:p><jats:bold>Conclusions:</jats:bold> Our results suggest that dysregulated expression of <jats:italic>Bcl2</jats:italic> and <jats:italic>Bax</jats:italic>, which leads to a high <jats:italic>Bcl2/Bax</jats:italic> ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL.</jats:p>	[ { "section_content": "Uvod: Rezistencija na apoptozu koja karakteri{e maligne B limfocite in vivo u hroni~noj limfocitnoj leukemiji (HLL) delimi~no je uzrokovana unutra{njim poreme}ajima apoptotske ma{inerije u ovim }elijama.Ti poreme}aji su rezultat geneti~kih promena i aberantne ekspresije regulatora procesa apoptoze, me\|u kojima klju~nu ulogu imaju ~lanovi Bcl2 familije.Cilj: Cilj ove studije je bio da se ispita udru`enost nivoa ekspresije proapoptotskog Bax gena, kao i Bcl2/Bax odnosa, sa klini~kim karakteristikama bolesnika sa HLL kao i molekularnim prognosti~kim markerima, i to mutacionim statusom rearan`iranih gena za te{ke lance imunoglobulina (IGHV) i ekspresijom gena za lipoproteinsku lipazu (LPL).Metode: Analizirana je ekspresija Bax iRNK i Bcl2/Bax iRNK odnos u mononuklearnim }elijama periferne krvi 58 bolesnika sa HLL i 10 zdravih kontrola metodom reverzne transkripcije i lan~ane reakcije polimeraze u realnom vremenu (qRT-PCR).Rezultati: Detektovana je povi{ena ekspresija Bax gena u HLL uzorcima u odnosu na kontrolne uzorke (p=0,003), kao i povi{en Bcl2/Bax odnos (p=<0,001).Kada je u pitanju udru`enost sa prognosti~kim markerima, Bcl2/Bax od nos je ispoljio negativnu korelaciju sa vremenom udvo -Abbreviations: CLL, chronic lymphocytic leukemia; Bcl2, Bcell lymphoma 2; Bax, Bcl2-associated X; IGHV, immu noglobulin heavy chain variable region genes; LPL, lipoprotein lipase; mRNA, messenger ribonucleic acid; miR, micro ribonucleic acid; PBMC, peripheral blood mononuclear cells; LDT, lymphocyte doubling time; LDH, lactate dehydrogenase; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; ROC, receiver operating characteristic; A, area under the ROC curve; CI, confidence interval. ", "section_name": "Kratak sadr`aj", "section_num": null }, { "section_content": "Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia in Europe and North America, affecting predominantly elderly individuals aged approximately 65-70 years at diagnosis.It is characterized by monoclonal expansion of circulating small, mature CD5 + CD19 + CD23 + sIgM low B lymphocytes.The most striking feature of CLL is its extremely variable clinical presentation, with diverse therapy requirements and overall survival.In some patients the disease can follow an indolent course for years without developing any symptoms, while in others rapid progression and need of treatment occur soon after diagnosis (1).This fact has led to an extensive search for new cellular and molecular prognostic markers which could predict the clinical course of CLL at the time of initial diagnosis and enable the development of risk-adapted therapeutic strategies (2)(3)(4). Circulating CLL B lymphocytes are arrested in G 0 /early G 1 phase of the cell cycle (5) and their gradual accumulation in blood, bone marrow and secondary lymphoid organs is being attributed primarily to de fective apoptosis.Although a growing body of evidence suggests that CLL clone turnover is more dynamic than previously assumed, and that CLL cells pro liferate and die at considerable rates (6, 7), impair ment of apoptosis remains one of the hallmarks of CLL.The fact that CLL cells undergo spontaneous apoptosis when placed in culture points to the role of microenvironment-derived signals in their survival in vivo (8).On the other hand, marked inter-patient variability in the rate of apoptosis of CLL cells ex vivo implicates the existence of inherent differences in their apoptotic po tential (9)(10)(11).Indeed, genetic alterations and aber rant expression of many apoptotic regulators involved in both intrinsic (mitochondrial) and extrinsic (death receptor) apoptotic pathways have been des cribed in CLL. Bcl2 family of proteins plays a central role in the regulation of the mitochondria-mediated pathway of apoptosis, and it is generally accepted that their deregulated function is involved in the pathogenesis and/or progression of CLL.Bcl2 family consists of pro-apoptotic (Bax, Bak, Bok, Bim, Bad, Bid, Bik, Bmf, Hrk, Noxa, Puma), and anti-apoptotic proteins (Bcl2, Bcl-X L , Mcl-1, A1, Bcl-W) (12).Given the functional antagonism between the pro-and anti-apoptotic Bcl2 family members, it is believed that the ratio of their activity levels is a critical determinant of the cells' susceptibility to apoptosis, rather than the levels of individual proteins. Leukemic cells of the majority of CLL patients overexpress the anti-apoptotic Bcl2 gene even though translocation (14;18), which juxtaposes Bcl2 to the immunoglobulin heavy chain enhancer, is a very rare event in CLL (13,14).It has been suggested that the main mechanism underlying Bcl2 upregulation is hypomethylation of its promoter region, detected in a large proportion of patients (15).In addition, miR-15a and miR-16-1, which negatively regulate Bcl2 at the posttranscriptional level, are frequently downregulated or lost by the deletion of 13q14, the most com mon genomic aberration in CLL (16,17).Abnormal ex pres sion of other Bcl2 family members has also been observed in CLL, namely Mcl1, BclX L , Bag-1, Bax, Bak, Bad (18)(19)(20), as well as Bcl2L12 and Bfl-1 (21,22).However, the results of different studies regarding the relationship between the expression of Bcl2 family genes and proteins and the disease stage, clinical progression and response to treatment are highly discrepant. In our previously published paper, we reported a significant overexpression of Bcl2 in a cohort of CLL patients compared to non-leukemic controls, and association of high Bcl2 mRNA levels with adverse prognostic parameters: progressive CLL, high serum b2-microglobulin, shorter lymphocyte doubling time (LDT) and high lipoprotein lipase gene (LPL) expression (21).In the study presented here, we broadened our previous research by analyzing the expression of the pro-apoptotic Bax gene in the same cohort of patients.The aim was to evaluate the association of Bax mRNA levels, as well as Bcl2/Bax ratio, with clinical and molecular prognostic markers in CLL, namely the mutational status of rearranged immunoglobulin heavy chain variable region genes (IGHV) and lipoprotein lipase gene expression.IGHV mutational status is the most powerful and the most stable molecular marker in CLL.Unmutated IGHV rearrangements represent an adverse prognostic factor and are was associated with LPL-positive status (p=0.035).Both the expression of Bax and Bcl2/Bax ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance.Conclusions: Our results suggest that dysregulated expression of Bcl2 and Bax, which leads to a high Bcl2/Bax ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL. Keywords: apoptosis, Bax, Bcl2/Bax ratio, chronic lymphocytic leukemia, expression analysis stru~avanja broja limfocita (r=-0,307; p=0,0451), dok je visoka ekspresija Bax bila povezana sa LPL-pozitivnim statusom (p=0,035).I ekspresija Bax gena i Bcl2/Bax odnos su bili vi{i kod bolesnika sa nemutiranim u odnosu na bolesnike sa mutiranim IGHV genima, ali nije dostignuta statisti~ka zna~ajnost.Zaklju~ak: Rezultati ove studije ukazuju na mogu}u ulogu poreme}ene ekspresije Bcl2 i Bax gena, koja dovodi do visokog Bcl2/Bax odnosa u leukemijskim }elijama, u patogenezi i klini~kom toku HLL. Klju~ne re~i: analiza ekspresije, apoptoza, Bax, Bcl2/Bax odnos, hroni~na limfocitna leukemija associated with shorter time to progression and overall survival (23)(24)(25).Lipoprotein lipase (LPL) is a novel molecular marker whose high-level expression is associated with unfavourable prognostic parameters in CLL, and which has been proposed as a surrogate marker for the IGHV mutational status (26)(27)(28). ", "section_name": "Introduction", "section_num": null }, { "section_content": "This study enrolled a total of 58 unselected patients from the Hematology Clinic, Clinical Cen ter of Serbia (Belgrade, Serbia), diagnosed as typical B cell CLL based on the clinical criteria and laboratory features.The study was approved by the medical ethic committee of the institution. The patient group consisted of 45 men and 13 women (male/female ratio = 3.5), with a median age of 63.5 years (range 39-86) at the time of diagnosis.Median white blood cell count was 55×10 9 /L (range 13.5-413), and median lymphocyte count was 42×10 9 /L (range 4.1-371).The distribution of clinical Binet stages was as follows: 22 patients (42.3%) stage A, 7 patients (13.5%) stage B and 23 patients (44.2%) stage C (the staging information was unavailable for 6 patients).Lymphocyte doubling time (LDT) was determi ned in 43 out of 58 patients; LDT ranged from 1 to 84 months, with a median of 12 months.Among 52 patients for whom we possessed follow-up information, progressive disease was observed in 40 patients (76.9%), whereas non-progressive disease was observed in 12 patients (23.1%).Patients were considered to have progressive disease based on at least one of the following criteria: lymphocyte doubling time of less than 1 year, progression to a more advanced Binet stage, development of systemic symptoms or Richter syndrome, or a downward trend of hemoglobin/platelet count to below the normal range. Serum markers b2-microglobulin and lactate dehydrogenase were determined in 32/58 and 37/58 patients, respectively.The levels of b2-microglobulin ranged 0.21-13.5 mg/L, with a median of 3.86 mg/L.Twenty-five (67.6%) patients had normal levels of LDH, while in the remaining 12 patients (32.4%)LDH was elevated.CD38 expression, lipoprotein lipase expression and IGHV mutational status were determined as reported in Karan-Djurasevic et al. (21).CD38 ex pression was assessed in 38 out of 58 CLL samples and, applying the cut-off level of 30% of CD38 positive cells, 14 patients (36.8%) were classified as CD38positive, and 24 patients (63.2%) as CD38-ne gative.Regarding IGHV mutational status, 29 of our patients (50%) belonged to the mutated CLL subset (M-CLL), while the other 29 patients (50%) belon ged to the unmutated CLL subset (U-CLL). In all patients who received treatment (40/58) no therapy had been administered for at least 6 months prior to blood sampling.The control group consisted of 10 healthy individuals, 3 men and 7 women, with a median age of 53 years (range 44-84). Peripheral blood mononuclear cells (PBMC) of all patients contained >90% of CLL lymphocytes, as confirmed by immunophenotyping.PBMC were isolated by Ficoll density-gradient centrifugation and total RNA was extracted using TRI reagent (Sigma-Alldrich).The isolated RNA was reverse-transcribed using RevertAid M-MuLV Reverse Transcriptase (Fermentas) and random hexamer primers, according to manufacturer's instructions. Bax mRNA expression was analysed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using SYBR Green chemistry in a 7500 Real Time PCR system (Applied Biosystems).The specific primers used for qRT-PCR amplification were: forward 5'-TGGCAGCTGACATGTTTTCTGAC-3' and reverse 5'-TCACCCAACCACCCTGGTCTT-3'.The amplification of Abl using the following primers: forward 5'-TGGAGATAACACTCTAAGCATAACTAAAG-GT-3' and reverse 5'-GACGTAGTTGCTTGGGACC-CA-3', served as internal control.The reaction mix ture contained 50 ng cDNK, 1 × Power SYBR® Green PCR Master Mix (Applied Biosystems) and 0.5 pmol (Bax) or 2 pmol (Abl) of each gene-specific primer, in a final reaction volume of 10 mL.The cycling conditions were as follows: denaturation of the template at 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute.Each qRT-PCR reaction was performed in duplicate, in order to evaluate reproducibility of the results.Quantification of target gene expression was made by a comparative ddCt method, using HL-60 cell line as the calibrator. ", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "Statistical analyses were performed using Fisher's exact test, Mann-Whitney rank-sum test, Spearman rank order correlation and receiver operating characteristic (ROC) analysis.All statistical tests were carried out using Sigma Stat 3.5 and SigmaPlot 11.0 software (Systat Software Inc.).Statistical significance was defined as p<0.05. ", "section_name": "Statistical analysis", "section_num": null }, { "section_content": "In this study, we analyzed the expression of Bax gene and Bcl2/Bax ratio in a cohort of 58 unselected patients with chronic lymphocytic leukemia. ", "section_name": "Results", "section_num": null }, { "section_content": "qRT-PCR expression analysis of Bax revealed significantly higher levels of Bax mRNA in CLL samples in comparison to non-leukemic samples (p=0.003;Mann-Whitney rank sum test).However, the patientto-patient variability and the increase of expression level in CLL vs. healthy controls was less prominent than in the case of Bcl2 (Figure 1). The expression of Bax did not show association with gender and Binet staging.Although we observed a tendency towards a negative correlation between Bax expression and the age at diagnosis, statistical significance was not reached (r=-0.26;p=0.0683;Spearman rank order correlation).Bax mRNA level was not associated with the course of the disease (progressive vs. non-progressive) and LDT.In addition, neither correlation to the levels of serum markers b2-microglobulin and LDH, nor association with CD38 status were detected. Bax was expressed at higher levels in U-CLL vs. M-CLL, but the difference in Bax expression between these two groups of patients did not reach statistical significance (p=0.056;Mann-Whitney rank sum test). In order to investigate the relationship between LPL expression and the expression of Bcl2, Bax and Bcl2/Bax ratio, we used median LPL mRNA expression as a cut-off level to define LPL status.According to this cut-off level, 29 patients (50%) were LPL-positive and 29 patients (50%) LPL-negative.In addition, LPL-positive status showed strong association with unmutated IGHV genes (p<0.001;Fisher's exact test), with only 6 discrepant cases (10.3%). Having divided our cohort into two groups based on LPL status, we then used median expression of Bcl2 and Bax as a cut-off level to discriminate between high and low expressing cases.By applying this approach, we found that high levels of both Bcl2 and Bax expression were associated with an LPL-positive status (p=0.008 and p=0.035, respectively; Fisher's exact test). ", "section_name": "Bax expression", "section_num": null }, { "section_content": "In our cohort of patients, Bcl2 and Bax expression levels were positively correlated (r=0.6;p=0.00;Spearman rank order correlation).The ratio of Bcl2 and Bax mRNA expression (Bcl2/Bax ratio) was significantly higher in CLL samples in comparison to healthy controls (p<0.001;Mann-Whitney rank sum test) (Figure 2).Bcl2/Bax ratio was not found to be significantly associated with either gender, Binet stage or the course of the disease.Similar to Bax expression, the observed trend toward negative correlation to the age at diagnosis was not statistically significant (r=-0.265;p=0.0627;Spearman rank order correlation).On the other hand, there was a significant negative correlation between Bcl2/Bax ratio and LDT (r=-0.307;p=0.0451;Spearman rank order correlation).No association with the levels of b2-microglobulin, LDH and CD38 status was observed.Bcl2/Bax ratio was higher in U-CLL vs. M-CLL and LPL-positive vs. LPL-negative groups of patients, but the association with either IGHV mutational status or LPL status was not statistically significant. We performed receiver operating characteristic (ROC) analysis in order to evaluate the discriminatory power of Bcl2 and Bax mRNA expression in CLL.ROC analysis demonstrated that both Bcl2 expression and Bcl2/Bax ratio efficiently distinguished CLL from non-leukemic samples (A=0.98,95% CI=0.95-1.009,qRT-PCR analysis showed a significantly higher Bcl2/Bax mRNA ratio in mononuclear cells of CLL patients in comparison to healthy controls.p<0.0001 and A=0.96, 95% CI=0.9230-1.005,p<0.0001, respectively), while Bax expression was found to be less discriminating (A=0.80,95% CI=0.6920-0.9097,p=0.002514) (Figure 3). ", "section_name": "Bcl2/Bax ratio", "section_num": null }, { "section_content": "Chronic lymphocytic leukemia is considered to be a paradigmatic example of malignancy caused by dysregulation of apoptosis.However, the unique mech anism preventing CLL cells from undergoing apoptosis in vivo is still elusive, as is the significance of their apoptotic resistance for the clinical course of the disease.Impairment of apoptosis results from the combination of microenvironmental survival signals and inherent genetic and epigenetic alterations of apoptotic machinery in CLL cells, both of which exert high patient-to-patient heterogeneity.Abnormalities in different apoptotic pathways have been described in CLL, namely ATM-p53 pathway (29)(30)(31), PI3K/Akt pathway (32)(33)(34), NF-kB pathway (32,35) and Fas/FasL system (36,37).Aberrant expression and genetic changes of Bcl2 family members, the key regulators of the intrinsic apoptotic pathway, have also been implicated in CLL (38).In addition, various cytokines, notably BAFF (»B-cell activation factor«), APRIL (»a proliferation inducing ligand«), CD40 lig-and and interleukin 4, promote survival of CLL cells in both a paracrine and autocrine manner (39)(40)(41).Finally, it is noteworthy that the activation of B cell receptor (BcR) also affects apoptotic pathways, although responsiveness of CLL cells to antigenic stimulation and signalling via surface IgM and IgD still remain controversial (42).Even though dysregulation of apoptosis is a distinctive feature of CLL, none of the apoptotic defects has been found to be universally present among CLL patients.This may, at least in part, explain the heterogeneity of the clinical course and response to therapy in CLL. In our previous research, we investigated the expression of Bcl2 gene, a prototypical anti-apoptotic member of the Bcl2 family, and detected a significant overexpression of Bcl2 in CLL samples, as well as association of high Bcl2 mRNA levels with un favourable prognostic markers (21).In the present study, we continued our research by analyzing the expression of Bax, a functional anta gonist of Bcl2, and the Bcl2/Bax ratio in CLL patients.The level of Bax expression we measured was significantly increased in CLL samples, but it was less heterogeneous and more overlapping with that of healthy controls than in the case of Bcl2.Given its pro-apoptotic role, the overexpression of Bax in CLL cells may seem paradoxical but, interestingly, an overexpression of both pro-and anti-apoptotic proteins has been observed in CLL (43).Moreover, their relative expression levels were positively correlated (44,45).This is considered to be a compensatory mechanism used by cells in an attempt to regain equili brium between proand anti-apoptotic proteins, which is crucial for apoptosis regulation. Although in some studies higher expression of Bax mRNA and protein was detected in non-progressive vs. progressive CLL (46,47), in our cohort no association with the course of the disease, Binet stage or LDT was observed.Regarding the association of Bax with the molecular prognostic markers, we observed higher Bax mRNA levels in U-CLL vs. M-CLL patients but, as was the case with Bcl2, without reaching statistical significance.On the other hand, high expression of both Bcl2 and Bax was associated with an LPL-positive status which, in turn, was an excellent predictor of unmutated IGHV genes.Several studies that investigated differential gene expression in U-CLL vs. M-CLL did not detect a significant association between Bcl2 and Bax expression and the IGHV mutational status (48,49).However, our results show high Bcl2 and Bax expression in the group of patients defined by LPL positivity and unmutated IGHV rearrangements.In a study of Pallasch et al. (50), it was demonstrated that the LPL inhibitor orlistat has a cytotoxic effect on primary CLL cells through specific and concentration-dependent induction of apoptosis.In addition, the authors found that BcR stimulation significantly increases LPL expression in CLL cells.Thus, it would be important to elucidate the mecha- Bcl2 mRNA expression and Bcl2/Bax mRNA ratio exert very high discriminatory power between CLL patients and healthy controls.Bcl2: A=0.98, sensitivity=0.95,specificity=1.00,95% CI= 0.95-1.009,p<0.0001Bcl2/Bax ratio: A=0.96, sensitivity=0.90, specificity=1.00,95% CI=0.9230-1.005,p<0.0001Bax: A=0.80, sensitivity=0.79,specificity=0.90, 95% CI= 0.6920-0.9097,p=0.002514 nisms by which intracellular pathways of lipid metabolism, apoptotic and BcR signalling are interconnected in CLL. Analysis of the Bcl2/Bax mRNA ratio revealed that it was higher in CLL samples in comparison to control samples and that it efficiently discriminated patients from healthy controls.However, the Bcl2/Bax ratio was not significantly related to either clinical characteristics of CLL (with the exception of LDT), or molecular prognostic markers, although it was slightly higher in LPL-positive vs. LPL-negative and U-CLL vs. M-CLL groups of patients.According to the results of some previous studies, an elevated Bcl2/Bax ratio, measured at both the mRNA (46) and protein level (47), is associated with adverse prognostic parameters in CLL and is more relevant for the survival of CLL cells than the expression levels of Bc2 and Bax individually.In other studies, including ours, however, this association was not observed (43).The explanation for these contradictory results may lie in the fact that other members of the Bcl2 family modulate the function of Bcl2 and Bax and, hence, their relative expression and/or activity levels also affect the CLL cells susceptibility to apoptosis.For example, the antiapoptotic protein Mcl1 is overexpressed in CLL and its high expression has been linked with poor prognosis in CLL patients (43,51,52).Like Bcl2, Mcl1 forms heterodimers with Bax, so elevated expression of Bcl2 and Mcl1 has an additive or synergistic negative effect on the Bax's pro-apoptotic function.Saxena et al. (51) suggested that, when it comes to response to apo ptosis-inducing chemotherapeutics, a negative effect of high Bcl2/Bax ratio may be overcome by low Mcl1 expression. In summary, the results of this study further emphasize the complexity of the role that the dysregulated expression of Bcl2 family members plays in prolonged survival of CLL cells and phenotype of the disease.Considerable inter-patient variability in their expression may contribute to the heterogeneity of CLL, but the association with clinical characteristics and molecular prognostic markers remains controversial.Further studies are needed to clarify to what extent the pattern of expression and/or activity of Bcl2 family genes and proteins influences the clinical behaviour of CLL. ", "section_name": "Discussion", "section_num": null } ]	[ { "section_content": "Acknowledgement.This work was supported by grant No. III 41004, Ministry of Education and Scien ce, Republic of Serbia. ", "section_name": "", "section_num": "" }, { "section_content": "The authors stated that they have no conflicts of interest regarding the publication of this article. ", "section_name": "Conflict of interest statement", "section_num": null } ]
10.1371/journal.pone.0134706	Interleukin 21 Controls mRNA and MicroRNA Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells	Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes), whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process.	[ { "section_content": "B-cell chronic lymphocytic leukemia (CLL) is a common type of leukemia, characterized by the progressive accumulation of CD5+ monoclonal B lymphocytes in peripheral blood, bone marrow and lymphoid tissues [1,2].The expansion of the CLL clone is due to an imbalance between cell death and proliferation [3].Clonal expansion occurs in specific niches within the lymphoid tissues and the bone marrow where CLL cells are protected from apoptosis [4,5].In this supportive microenvironment, CLL cells establish interactions with multiple cell types, including activated CD4+ T cells expressing CD40 ligand (CD40L) [6].In addition, antigenic stimulation is involved in CLL cell activation and proliferation via the triggering of their B-cell receptor (BCR) complex, and evidence from several studies indicate that CLL cells derive from antigen-experienced B-cells [7][8][9].Besides CD40L and the antigen, several other molecules regulate CLL survival and proliferation.For example, nurse-like cells and stromal endothelial cells support the survival of CLL cells in vitro through contact-dependent stimuli, mediated by members of the tumor necrosis factor (TNF) superfamily [10,11].In addition, several chemokines and cytokines have been reported to regulate CLL cell survival and proliferation [5].For example, the chemokine CXC ligand 12 (CXCL12; also known as stromal cell-derived factor-1, SDF-1), which is produced by nurse-like cells [12], mediates anti-apoptotic effects in CLL cells via the CXC chemokine receptor type 4 (CXCR4).Importantly, chemokines have also been involved in orchestrating the crosstalk between CLL cells and their supportive cells within the microenvironment.Thus, CC ligand 3 (CCL3) and CCL4 are produced by CLL cells undergoing BCR stimulation or co-culture with nurse-like cells [13].In turn, these factors attract CC receptor type 1 (CCR1)-expressing monocytes/macrophages, which activate endothelial cells to support CLL cell survival [14].In addition, CLL cells produce CCL22 and CCL17 in response to CD40L stimulation and CCL22 attracts CCR4+CD4+CD40L+ T cells, which further stimulate CLL cells [15]. Among the cytokines, hepatocyte growth factor (HGF), which is produced by different types of mesenchymal cells, supports CLL cell survival [16].In addition, cytokines of the interleukin (IL) 2 family, such as IL4 and IL15, mediate CLL cell survival and proliferation [17,18].In contrast, IL21, a regulator of normal B-cell survival [19] and differentiation, [20] was shown to induce CLL B-cell apoptosis [21][22][23].These data were obtained using recombinant IL21 at pharmacological dosages (50-100 ng/ml).However, IL21 produced by CD4+CD40L+ T cells supported the proliferation of co-cultured CLL cells [24], acting in concert with other T-follicular helper-derived cytokines such as IL4 [25].Another report suggested that IL21, in combination with toll-like receptor (TLR) 9 agonists, exerts differential effects on CLL cells from progressing or non-progressing patients [26].IL21 may also induce CLL B-cell differentiation through the induction of B-lymphocyte-induced maturation protein-1 (Blimp-1), a regulator of plasma cell induction [27]. Moreover, IL21 also mediates the apoptosis of various non-Hodgkin's lymphomas, including follicular [28], mantle cell [29] and diffuse large B-cell lymphoma [30].The observation that IL21 inhibits the survival of some neoplastic B-cells may have translational implications.Indeed, a phase I dose-finding trial of IL21 and rituximab in relapsed and refractory low-grade B-cell malignancies suggested clinical activity [31].Several lines of evidence indicate that IL21-mediated effects require gene transcription through the JAK3/STAT-1 and -3 pathways, which mediate apoptosis in sensitive cells [19,[21][22][23][28][29][30].Other cytokines, such as interferons, may regulate gene expression through the modulation of specific miRNAs, which target specific mRNAs [32,33]. To gain additional knowledge on the molecular mechanisms of IL21 activity on neoplastic B-cells, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells.An integrated analysis of miRNA and mRNA expression suggested a role of miRNAs in the regulation of gene expression by IL21. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Materials and Methods", "section_num": null }, { "section_content": "This study was approved by the institutional review board of Istituto Nazionale per la Ricerca sul Cancro (Genoa, Italy) and by the Ethical Committee of Regione Liguria.Peripheral blood samples were obtained from untreated CLL patients (Table 1) upon written informed consent, according to our institutional procedure and the Declaration of Helsinki.B-cell populations were enriched from peripheral blood mononuclear cells by negative selection with antibodycoated magnetic beads (anti-CD2; Dynal-Invitrogen, Oslo, Norway) to consist of >95% of CD19+/CD5+ cells.The B-cell RosetteSep kit (Stemcell Technologies, Grenoble, France) was used to isolate normal B-cells from buffy coats of six age-matched healthy donors.CLL or normal B-cells were then pre-activated by co-culture with adherent, irradiated CD40L-expressing murine fibroblasts for 36-48 h, collected and stimulated for an additional 18 h with either medium only or recombinant human IL21 produced in E. coli (Biosource-Invitrogen, San Diego, Ca) at 80 ng/ml as described [21].The CD40L-expressing NIH-3T3 murine fibroblast cell line, stably transfected with human CD40L cDNA, was kindly provided by Franco Fais (Genova, Italy) and was generated as previously described [34].A dose of 80 ng/ml was used, based on dose-response analyses.CD40L pre-stimulation allowed the enhancement of IL21 receptor (IL21R) expression on CLL cells and their sensitization to IL21 activity [21].Apoptosis was assessed by fluorescence-activated cell sorting (FACS) analysis of annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining (Bender, Vienna, Austria). ", "section_name": "CLL and normal B-cell isolation, activation and apoptosis detection", "section_num": null }, { "section_content": "Total RNA was isolated from both untreated and IL21-stimulated CLL and normal B cells, using TRIzol (Invitrogen-LifeTechnologies, Carlsbad, CA, USA).Total RNA from 13 paired untreated and IL21-stimulated cells was then amplified in vitro, labeled with biotin and hybridized on Sentrix Bead Chip HumanRef_8_v2 (Illumina, San Diego, CA, USA).Array chips were washed, stained with 1 mg/ml of Cy3-streptavidine (GE Healthcare, Pittsburgh, PA) and scanned with Illumina BeadArray Reader (Illumina, San Diego, CA, USA).For each gene, a detection threshold with a p value < 0.05 was set, and 50% of missing values were allowed as a cutoff to filter the reliable data, yielding an expression matrix containing 12,166 probes.In addition, nine pairs of samples were miRNA-profiled on Illumina Human miRNA_v2 chips.Mature miRNAs were amplified, fluorescently labeled, hybridized on Illumina miRNA Bead-Chips and analyzed by the Illumina BeadArray Reader, as described previously [35].Primary data were collected using BeadStudio V3.0 software (Illumina, San Diego, CA, USA).All microarray data were compliant to MIAME (Minimum Information About a Microarray Experiment) guidelines and were deposited into the Gene Expression Omnibus (GEO) database of NCBI (National Center for Biotechnology Expression) (http://www.ncbi.nlm.nih.gov/geo/), with accession numbers GSE42158 and GSE42160. ", "section_name": "Microarray analysis", "section_num": null }, { "section_content": "TaqMan gene primer sets were purchased from Life Technologies (LifeTechnologies, Carlsbad, CA, USA).Reverse transcription and PCR amplification for gene expression assays were performed with TaqMan Transcription and TaqMan Gene Expression Master Mix, respectively, following the manufacturer's instructions (LifeTechnologies, Carlsbad, CA, USA).RT-qPCR data were normalized, using POL2RA as housekeeping gene.miRNA RT-qPCR was performed using the miRCURY LNA Universal RT microRNA PCR system (Exiqon, Vedbaek, Denmark) according to the manufacturer's instructions. Total RNA (20 ng) was polyadenylated and reverse-transcribed at 42°C (60 min), in a reaction volume of 20 μl using a poly-T primer containing a 5 0 universal tag.After heat-inactivation at 85°C the resulting cDNA was diluted 80-fold in nuclease free water, and a volume of 8 μl was amplified in 20 μl reaction volume as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 10 s and 60°C for 60 s.Normalization was performed with SNORD48 [36]. Gene and miRNA expression levels were quantified, using a sequence detection system (ABI Prism 7900HT; LifeTechnologies, Carlsbad, CA), and the threshold cycle (Ct) for each sample was determined.ABI SDS 2.4 software (LifeTechnologies, Carlsbad, CA, USA) was used to recover the data, and the relative expression was calculated, using the comparative ΔCt method. ", "section_name": "Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analysis", "section_num": null }, { "section_content": "Supernatants from either untreated or IL21-treated CLL cells were harvested 36 h post stimulation and assayed by ELISA for CCL3, CCL4, CCL17, CCL22, CXCL9 and CXCL10 according to the manufacturer's protocol (R&D Systems, Minneapolis, MN, USA). ", "section_name": "Enzyme-linked immunosorbent assay (ELISA) for chemokines", "section_num": null }, { "section_content": "Hsa-miR-663b, its specific antagonist or an irrelevant synthetic RNA (Qiagen, Milan, Italy) were transfected, in triplicate, in CD40-activated CLL cells (10 6 cells) at 50 nmol/l, using the AMAXA nucleofection system, the human B-cell kit and the U-015 program (Lonza, Cologne, Germany).After 18 h of culture, RNA was extracted for mRNA analysis. ", "section_name": "miRNA and antagomir transfection", "section_num": null }, { "section_content": "Differentially expressed genes were defined according to the following criteria: false discovery rate (FDR) <0.01 and a fold-change threshold >1.5 or < 0.667.The open-source MultiExperiment Viewer v4.6 was used for principal component analysis (PCA), and the BrB Array-Tools_v4.1.0-stablerelease (http://linus.nci.nih.gov/brb) was used for class comparison, hierarchical clustering, global test and heat map analyses.In this study, we used weighted gene co-expression network analysis (WGCNA) [37] to determine the gene co-expression networks regulated by IL21 in CD40-activated CLL cells from microarray gene expression data. WGCNA is a systems biology method that enables the description of the correlation patterns among genes and the identification of modules of highly correlated genes across microarray samples.As a result, WGCNA offers a meaningful data reduction scheme, focusing the analysis on modules expected to correspond to specific biological functions.The method is based on the computation of pair-wise Pearson correlations between each gene pair, allowing the assessment of the topological overlap (TO), a measurement of the relationships of the two genes against all other genes.A hierarchical map, using 1-TO as the distance measure, was built to cluster the genes, and a tree-cutting algorithm was used for module assignments.The module eigengene (ME) is a representative value of each module, which is equivalent to the first principal component and explains the largest proportion of variance of the module genes.Submap analysis was performed, using subclass mapping [38], to assess the degree of molecular correspondence among different datasets.IPA 8.5 (Ingenuity Pathway Analysis, Ingenuity Systems; Qiagen Venlo, The Netherlands) was used to analyze the functional and network connections. Integration of miRNA and mRNA data was performed, using the MAGIA tool [39], and visualized by Cytoscape [40] miRNA, gene expression and protein levels were compared, using a two-sample paired Student's t-test with random variance model. ", "section_name": "Statistics and bioinformatic analyses", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "Under the experimental conditions used, IL21 induced a slow-rate apoptotic process, which became clearly evident at 48-96 h, while no significant apoptosis was induced by IL21 at 18 h when compared to untreated cells (Fig 1).IL21 induced apoptosis (>10% of induction relative to untreated cells) in 40% of cases, and no correlation between apoptosis and CLL clinical and biological characteristics was evident. Then we studied how IL21 could modify gene expression by means of high-throughput analysis of CLL cells from a first cohort of 13 patients (Table 1).A large number of genes were modulated following IL21 treatment, as represented by the volcano plot (Fig 2A).According to the imposed thresholds, a total of 563 differentially expressed genes (corresponding to 582 probes) were retrieved, among which 275 genes were up-regulated and 288 down-regulated.PCA showed that the samples were distributed in two main clusters, matching the IL21-treated and -untreated CLL cells (Fig 2B).In this cohort, the pattern of IL-21-regulated genes was not significantly different in subgroups stratified on the basis of IgVH mutational status or of ZAP70 or CD38 expression. Submap analysis [39] of four publicly available datasets of IL21-treated vs. untreated (i) human CLL cells (GSE50572) [24] (ii) human Sezary T cells (GSE8685) [41] (iii) murine naïve CD8+ T cells (GSE2059) [42] and (iv) murine pre-activated CD4+ T cells (GSE19198) [43] showed regulation of the same or orthologous genes coherent with those identified in our dataset (S1 Fig) .The dendrogram obtained by the WGCNA method showed that the differentially expressed genes clustered into three modules (defined as ME 'color' module), representing correlated networks of genes within each module (Fig 2C).To determine potential relationships among the modules, a set of seed \"eigengenes\", one for each module, was computed.The heatmap of the three module \"eigengenes\" (MEs) indicated a high level of correlation between the green and blue modules and, to a lesser extent, to the pink module (Fig 2D).The genes entering into each module are listed in S1 Table along with FDR and fold-change. Functional analyses of signaling pathways and network connections were performed by IPA.S2 Table shows the top functions (imposing a score of > 30) associated with each module.In the largest module (MEpink module) that included 272 genes, the three top functions were: (i) lipid metabolism, small molecule biochemistry, hematological system development and function, (ii) humoral immune response, protein synthesis, cellular development and (iii) endocrine system disorders, gastrointestinal disease, immunological disease (S2 Table ).Indeed, several modulated genes encoded cytokine receptors (IL2RA and IL2RB up-regulated; IL13RA1 down-regulated), cytokines (IL12A and TNFSF10 up-regulated; EBI3, IL15, TNF and TNFSF4 down-regulated) and chemokines (CCL17 and CCL22 down-regulated).The three most significant molecular pathways contain TNF, nuclear factor kappa B (NF-κB) or major histocompatibility complex (MHC) class II as hubs (S2 Table ).In the MEgreen module that included 206 genes, the four top functions were: (i) RNA posttranscriptional modification, cellular assembly and organization, cellular function and maintenance, (ii) RNA post-transcriptional modification, infectious disease, cardiovascular disease, (iii) cell-to-cell signaling and interaction, cellular movement, hematological system development and function, and (iv) carbohydrate, lipid and nucleic acid metabolism (S2 Table ).These networks suggest that IL21 may profoundly affect the metabolism of CLL cells.Networks 1 and 3 had the phosphoinositide 3 kinase (PI3K) and NF-kB complexes as respective hubs (S2 Table andFig 3).Interestingly, network 3 included the pro-inflammatory chemokines CCL3, CCL4 and CCL3L1/LOC730422, as well as the cytokine genes LTB and TNF, all down-regulated in IL21-treated CLL cells.In contrast, CXCL9 and CXCL10 chemokine genes were among the genes mostly up-regulated by IL21.In addition, the MEgreen module contained MYC and MYC-associated genes, which were all up-regulated by IL21 (S2 Table ). The MEblue module included 85 genes that were mainly up-regulated (84%) by IL21.The three top functions were: (i) DNA replication, recombination and repair, cell cycle, cancer, (ii) cancer, lipid metabolism, molecular transport, and (iii) cellular assembly and organization, cellular function and maintenance, post-translational modification (S2 Table ).Network 1 contained the highest number of up-regulated genes, including the transcription factor E2F1, the MCM (minichromosome maintenance) family members MCM-4, -5, -6, -7 and -10 and the growth arrest (GAS)-6 gene (S2 Table ). ", "section_name": "IL21 regulated gene expression in CD40-activated CLL cells", "section_num": null }, { "section_content": "In view of the number of chemokine genes modulated by IL21 and their role in CLL biology, the expression of these genes was confirmed in the same cohort and validated on a distinct set of 16 CLL cases (Table 2).RT-qPCR analyses confirmed that IL21 inhibited significantly the expression of CCL3, CCL3CL1, CCL4, CCL17 and CCL22 mRNA, relative to untreated cells, IPA analysis identified a functional network in the MEgreen module, corresponding to cell-to-cell signaling and interaction, cellular movement, hematological system development and function, with NF-κB as a hub.The network is displayed graphically as nodes (genes) and edges (the biological relationships between nodes).The node color intensity is related to the IL21-mediated changes in gene expression levels (red, up-regulated; green, down-regulated genes).doi:10.1371/journal.pone.0134706.g003while it up-regulated the Th1-related CXCL9 and CXCL10 chemokine gene expression.As a control, the chemokine CCL20, not differentially expressed in the arrays, was also assessed by RT-qPCR, confirming the absence of modulation by IL21.In addition, a group of biologically relevant genes were validated using RT-qPCR and showed consistent modulation by IL21 (Table 2).We then verified whether IL21 regulated chemokine protein production in a set of CLL cases (Table 1).The concentration of each chemokine was assessed by ELISA, in conditioned media of IL21-treated or untreated CLL cells.IL21 significantly inhibited CCL3, CCL4, CCL17 and CCL22, whereas it induced CXCL9 and CXCL10 secretion (Fig 4).Altogether, these data indicated that IL21 is a major regulator of chemokine expression in CD40-activated CLL cells. ", "section_name": "IL21 regulated the expression of chemokine mRNA and protein", "section_num": null }, { "section_content": "IL21 regulated a similar set of genes in normal CD40-activated B-cells isolated from the peripheral blood of age-matched healthy donors (Table 2).Most of the RT-qPCR-validated genes of the MEpink and MEgreen modules, including chemokine genes, were regulated in a similar fashion in CLL and normal B-cells.However, TNF and MYC genes, modulated in CLL cells, were unchanged in normal B-cells.Intriguingly, the three validated genes of the MEblue module E2F1, EGR2 and GAS-6 also showed no changes in normal B-cells.Thus, IL21 modulates the expression of several genes in both normal and CLL B-cells, although a set of genes is uniquely regulated in CLL cells. ", "section_name": "Gene-expression IL21 regulation in CD40 activated normal B-cells", "section_num": null }, { "section_content": "To investigate whether IL21 could regulate gene expressions through specific miRNAs, we analyzed whether IL21 modified the miRNome in CLL cells and performed an integrated analysis of miRNAs and gene expression profiles.Among the 565 miRNAs detected consistently in the nine sample pairs analyzed (Table 1), 63 miRNAs were modulated significantly by IL21 treatment.Thirty-nine of these miRNA were up-regulated, and 23 down-regulated, at p < 0.05, as shown by class comparison analysis (S3 Table ).Three representative miRNAs (up-regulated hsa-miR-663b, and down-regulated hsa-miR-125b-1 Ã and hsa-miR-708) were validated further by RT-qPCR, which confirmed the array data (S4 Table ).In normal B-cells, IL21 significantly modulated hsa-miR-663b but not hsa-miR-125b-1 Ã and hsa-miR-708 expression (S4 Table ).We compared the differentially expressed miRNAs to the differentially expressed genes in the same samples using the MAGIA software [39].Overall, 33 miRNAs (S5 Table) had a negative correlation with 290 of the 550 genes belonging to the modules.Nine miRNAs were potentially master regulators of 73% of genes differentially expressed, and their potential targets were distributed in all three modules.Among these miRNAs, hsa-miR-296-3p, hsa-miR-125b-1 Ã , hsa-miR-1225-5p and hsa-miR-1228 Ã showed the largest number of potential targets (S5 Table andFig 5).In contrast to these miRNAs, the potential target genes (27/28) of hsa-miR-663b clustered into the MEpink module (S5 Table ).The negatively correlated miRNA-mRNA interactions (S1 Table ) suggested the interplay between specific IL21-regulated miRNAs and potential target genes.The MAGIA tool clustered the negatively correlated miRNA/gene interactions into two main networks (Fig 5).The largest network included 17 IL21-up-regulated miRNAs, among which hsa-miR-296-3p, hsa-miR-663b and hsa-miR-1228 Ã showed the largest number of interconnected target genes.The other network included the IL21-down-regulated miRNAs hsa-miR-125b-1 Ã , hsa-miR-326, hsa-miR-542-5p and hsa-miR-652 and their inversely regulated target genes. ", "section_name": "Integrated analysis of miRNome and gene expression profiling indicated a potential role of miRNA in IL21-mediated gene regulation", "section_num": null }, { "section_content": "The miRNA hsa-miR-663b targeted specifically a set of genes belonging to a single module and pathway (S5 Table, S2 Fig) .As this finding suggested its involvement in a functional network encompassing cellular movement and hematological system development and function, we investigated this miRNA further.In particular, we focused our attention on its potential effect on CCL17, CD40 and PI3KCD mRNAs, which are biologically relevant to CLL, and on DDR1 mRNA that encodes a tyrosine kinase receptor expressed in several tumors and leukemias [44].To verify that hsa-miR-663b can affect gene expression, CD40L-activated CLL cells from five different patients were transfected with either hsa-miR-663b or an irrelevant RNA sequence.Conversely, IL21-stimulated CLL cells were transfected with either the hsa-miR-663b antagonist or the irrelevant RNA.An experimental control using RT-qPCR, showed that both hsa-miR-663b transfection and IL21 treatment strongly enhanced hsa-miR-663b intracellular levels in CLL cells (S3 Fig) .Transfection with the antagonist down-regulated hsa-miR-663b levels in RT-qPCR analyses showed that transfection with hsa-miR-663b down-regulated significantly the expression of CCL17, CD40, DDR1, and PIK3CD mRNAs in a similar fashion to IL21 (Fig 6).However, the hsa-miR-663b antagonist inhibited the down-regulation of these mRNAs in IL21-treated cells (Fig 6).As a control, we also tested the IL21-insensitive gene CCL20, which was not modulated by hsa-miR-663b or its antagonist (S4 Fig).These data indicated that IL21 modulated the expression of genes in CLL cells through mechanisms involving hsa-miR-663b and identified novel potential biological functions of this miRNA. ", "section_name": "Role of hsa-miR-663b in IL21-mediated gene regulation", "section_num": null }, { "section_content": "Our present findings indicate that IL21 can profoundly modify the mRNA and miRNA expression profiles in CD40-activated CLL cells.The gene and miRNA expression profiles were analyzed by microarray in a first cohort, and selected features were validated by qRT-PCR in the same case material and subsequently in an independent cohort.In addition, a coherent regulation of the same or orthologous genes was identified by submap analysis of a dataset of IL21-stimulated CLL cells [24] and three publicly available datasets of human and murine T cells stimulated by IL21 [41][42][43], suggesting a relevant role of the genes identified in our dataset.Finally, an integrated analysis of miRNA and mRNA expressions indicated that several miRNAs and mRNAs show significant anti-correlation, suggesting that IL21-modulated miR-NAs regulate gene expression in CLL cells.Although we used IL21 at concentration of 80 ng/ ml, which may represent a \"pharmacological\" dose, higher than physiological levels, our observations might be relevant considering potential therapeutic applications.In any case, the true physiological concentrations of IL21 in the local microenvironment of secondary lymphoid organs cannot be really established. Genes regulated by IL21 functionally clustered into different modules.The first module (MEpink) contained genes involved in hematological system development, humoral immune response and immune disease.Indeed, this cluster comprised several genes encoding cytokine receptors, cytokines and the chemokines CCL17 and CCL22.The two most significant functional networks of genes within this cluster had, as highly connected nodes, NF-κB and TNF, respectively. The NF-κB network included several down-regulated genes such as NFKBIZ, NFKBIE and FCER.FCER encodes FceR-II (CD23), which we reported previously as being down-regulated by IL21 [21].However, within the same network, STAT3 was up-regulated.IL21 induces STAT3 phosphorylation in CLL cells [21], and MYC is a known STAT3 target gene [45].Accordingly, MYC and other STAT3-dependent genes [46], such as STAT3, SOCS3, BCL3 and BCL6, were up-regulated by IL21 in CD40-activated CLL cells (S1 Table ).Previous findings indicate that STAT3 up-regulates c-Myc expression in IL21-treated diffuse large B-cell lymphoma [30].In this study, the STAT3/MYC pathway triggered apoptosis through the downregulation of the anti-apoptotic Bcl-2 and Bcl-X(L) proteins.Here, we also found that IL21 inhibited the expression of the anti-apoptotic genes BCL2 and BCL2L1 (the latter encoding Bcl-X(L) protein) in CLL cells, whereas it up-regulated the pro-apoptotic genes GZMB and BNIP3 (S1 Table ), in agreement with a previous report [24]. In addition, a previous study reported that IL21 up-regulates granzyme-B expression in CLL and normal B-cells, resulting in the acquisition of cytotoxic properties and the induction of cell apoptosis [22].In agreement with these data, GZMB was also up-regulated by IL21 in our study.Altogether, these data indicate that IL21 induces an imbalance in the expression of pro-and anti-apoptotic genes in CLL cells and suggest that IL21-mediated pro-apoptotic mechanisms are common to different B-cell neoplasms. Interestingly, not only TNF, but also the TNF-related LTB gene, which encodes lymphotoxin-β, was down-regulated by IL21.Lymphotoxin-β is expressed highly in CLL cells and may support CLL cell proliferation in vitro [47], suggesting that IL21 may interfere with autocrine loops involving lymphotoxin-β in CLL cells. The top functions of the second module (MEgreen) of IL21-modulated genes were RNA post-translational modification, cellular assembly and function, cell-to-cell signaling, cell movement and hematological system development.Pathway analysis showed PI3K and NF-κB complexes as highly connected nodes of two networks of regulated genes.Both PI3K and NF-κB are in biologically relevant pathways, which generate signals from several surface receptors, including the BCR complex and chemokine receptors, and mediate growth, survival and migration of CLL cells [48,49].Importantly, we found that IL21 down-regulated the expression of the PIK3CD gene, which encodes PI3Kδ.This PI3K isoform is activated constitutively in CLL cells, most likely through stimuli arising from the microenvironment [48,49].For this reason, PI3Kδ plays a crucial role in CLL biology and is considered a relevant target for therapy.Indeed, small molecule inhibitors of PI3Kδ, such as GS-1101, promote apoptosis of primary cells from CLL and other B-cell malignancies.In addition, CLL patients treated with GS-1101 showed a redistribution of CLL cells from the bone marrow and lymphoid organs in the periphery, and a reduction in CCL3 and CCL4 serum levels [49]. Importantly, in the present report, we showed that IL21 down-regulated the expression of the pro-inflammatory CCL3, CCL4 and CCL3L1 chemokine genes, and also those of CCL17 and CCL22, which are Th2-related chemokine genes.These data were confirmed further in independent cohorts and collectively indicate that IL21 inhibits the production of chemokines that favor the crosstalk of CLL cells with supportive cells within the microenvironment [13][14][15].In addition, IL21 up-regulated the expression of the Th1-related CXCL9 and CXCL10 chemokine genes, thus suggesting that IL21 mediates a shift from a Th2 to a Th1 chemokine profile in CLL cells.In murine tumor models, IL21 induces a Th1 immune response and antiangiogenic effects mediated by CXCL9 and CXCL10 [50].In general, most effects of IL21 on gene expression are similar in CLL and normal B-cells, indicating a role for these genes in normal B-cell responses.However, modulation of specific genes, such as TNF, MYC, E2F1, EGR2 and GAS-6, occurred only in CLL B-cells, suggestive of a specific role of IL21 in CLL biology.In particular, the EGR2 gene was recently found mutated in 8% of CLL cases with poor prognosis, suggesting a possible role of this gene in CLL pathogenesis, possibly through a deregulation of BCR intracellular signaling [51]. It is well known that specific miRNAs play an important role in CLL pathogeny and regulate the expression of several genes [52].In addition, some cytokines were reported to regulate different miRNAs [32][33].We therefore tested the hypothesis that specific sets of miRNAs could be modulated by IL21 and regulate gene expression in CLL cells.Indeed, our findings indicated that IL21 modulated significantly the expression of 63 miRNAs.To our knowledge, these miR-NAs were not implicated previously in CLL biology, and some of them have yet unknown functions.However, the present data may underestimate the number of IL21-modulated miRNAs, as the miRBase 12 chip allowed the detection of only about 50% of the known miRNAs (miR-Base 19).We then correlated miRNA to gene expression, using bioinformatic tools for data integration and prediction of potential miRNA/target gene interactions.The significant interactions involved 33 miRNAs, which were negatively correlated with 290 genes clustered in the different modules.Nine of these miRNAs appeared to regulate about 73% of expressed genes and could thus represent the main regulators of the effects of IL21 on CLL cells.In particular, we found that miR-125b-1 Ã , miR-1228 Ã , miR-296-3p and miR-1225-5p showed the largest number of potential interactions.Two of these miRNAs were 'star' sequences, which derive from pre-miRNA processing and were thought to be non-functional and rapidly degraded.However, this concept has been challenged, and several miR Ã , including miR-125b-1 Ã and miR-1228 Ã , have been renamed as hsa-miR-125b-1-3p and hsa-miR-1228-5p, respectively. To confirm further that miRNAs modulated by IL21 can affect gene expression, we selected hsa-miR-663b.This miRNA is one of the miRNAs mostly up-regulated by IL21 and is also related to a single functional cluster of genes (that is, the MEpink module).In addition, hsa-miR-663b showed an inverse relationship with the expression of potentially relevant IL21-regulated genes, suggesting its possible involvement in some IL21 effects.Indeed, CD40L-activated CLL cells transfected with hsa-miR-663b showed a significant down-regulation of CCL17, CD40, DDR1 and PI3KCD mRNAs.Conversely, the specific antagonist of hsa-miR-663b inhibited the down-regulation of the same genes mediated by IL21 treatment.Altogether, these data confirmed that IL21 can modulate the expression of genes in CLL cells through mechanisms involving the regulation of specific miRNAs and identify novel potential functions of hsa-miR-663b. Although hsa-miR-663b regulated the expression of four mRNA targets and had potential binding sites in the 3 0 UTR sequences, its activity might either be related to a direct action or involve indirect mechanisms through other targets.IL21 modulated hsa-miR-663b and its potential target genes not only in CLL, but also in normal B-cells, suggesting a general role of this miRNA in modulating both neoplastic and normal B-cell functions. In conclusion, our data showed that IL21 is a potent modulator of gene expression in CLL cells.IL21 may regulate gene expression not only through specific transcription factors, such as STAT3, but also through the regulation of several miRNAs that potentially target more than 200 genes. ", "section_name": "Discussion", "section_num": null } ]	[ { "section_content": "This work was funded by: AIRC (IG10492 to MF, IG13518 to SF); Special Program Molecular Clinical Oncology-\"5 per mille\", grant 9980, 2010-15 to MF; \"Innovative tools for cancer risk assessment and early diagnosis-5 per mille\", grant 12162 to SC; ", "section_name": "", "section_num": "" }, { "section_content": "All microarray data were MIAME-compliant and were deposited into the GEO (Gene Expression Omnibus) database of NCBI (National Center for Biotechnology Expression) (http://www.ncbi.nlm.nih.gov/geo/), with accession numbers GSE42158 and GSE42160. ", "section_name": "", "section_num": "" }, { "section_content": "S1 Fig. Subclass mapping analysis.Subclass mapping (SubMap) analysis comparing genomewide molecular patterns identified in the current study (A1 and A2) and those identified in publicly available microarray datasets (B1 and B2).Red color indicates high confidence for correspondence; blue color indicates lack of correspondance.P values are indicated in the corresponding boxes.GSE50572: IL21-treated vs untreated human CLL cells [24]; GSE8685: IL21-treated vs untreated human Sezary cells [41]; GSE2059: IL21-treated vs untreated murine naive CD8+ T cells [42]; GSE19198: IL21-treated vs untreated murine pre-activated CD4+ T cells [43].(PDF) S2 Fig. Ingenuity Pathway Analysis (IPA) of hsa-miR-663b targeted genes.The genes found as putative targets of hsa-miR-663b using MAGIA web-tool, identified a significant network (Infectious Disease, Cellular Movement, Hematological System Development and Function).In addition, IL21-stimulated CLL cells were transfected with the irrelevant RNA (irr IL21) or with hsa-miR-663b antagonist (a663b IL21).Expression was tested by RT-qPCR.Statistical analysis was performed by Kruskall-Wallis test.(PDF) S4 Fig. CCL20 expression in miRNA or antagomir-transfected CLL cells.CLL cells were transfected with an irrelevant RNA sequence (irr) or with hsa-miR-663b (663b).In addition, IL21-stimulated CLL cells were transfected with the irrelevant RNA (irr IL21) or with hsa-miR-663b antagonist (a663b IL21).Expression was tested by RT-qPCR.Statistical analysis was performed using the Kruskall-Wallis test.(PDF) S1 Table .Genes differentially expressed between IL21-stimulated and paired control CCL cells.genes belonging to modules and their anti-correlation with differentially expressed miRNA.(XLSX) S2 ", "section_name": "Supporting Information", "section_num": null } ]
10.1038/s41598-021-92412-8	Immunomodulatory effects of different intravenous immunoglobulin preparations in chronic lymphocytic leukemia	<jats:title>Abstract</jats:title><jats:p>Hypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.</jats:p>	[ { "section_content": "have been identified, for example: direct and indirect inhibition of T-cell activation 5 , induction of anergy and impairment of BCR-and TLR-signalling on B cells 6,7 , and inhibition of the mononuclear phagocytic system 8,9 . The immunomodulatory capacity of Ig preparations on CLL cells was not directly addressed until recently when Spaner, D. et al. showed that a SCIg preparation impaired BCR signaling, activation and cytokine secretion by CLL cells stimulated in vitro 10 .Interestingly, in that report they found that patients receiving IgRT that increases IgG levels over 9 g/L showed evidence of disease control, suggesting that high doses of Ig may have anti-leukemic activity in CLL patients. Because both, its particular isotype composition and the chemical treatments during manufacturing might affect the immunomodulatory capacity of an IVIg preparation, our aim was to explore in vitro the immunomodulatory capacity of Pentaglobin, an IVIg enriched in IgM/IgA (IVIgGMA) and Vigam, an IVIg preparation with more than 95% of IgG (IVIgG) in CLL.Given the capacity of IVIg to affect T cell compartment and the particular characteristics of T cells from CLL patients 11 , we extended our analysis not only to leukemic B cells but also to T lymphocytes. ", "section_name": "", "section_num": "" }, { "section_content": "The in vitro activation of T cells from CLL patients in response to TCR-stimulation is diminished by IVIgG but not IVIgGMA.Several reports have shown that IgG preparations decreased the activation of T cells from healthy subjects in vitro 5,12,13 .In order to evaluate whether IVIgGMA and IVIgG differentially regulate the activation of T cells from CLL patients, PBMC were stimulated in vitro with immobilized anti-CD3 mAb for 24 h, in the presence of IVIgGMA, IVIgG or HSA at equimolar concentration as control.Because previous reports showed that the inhibitory effect of IgG is observed at high concentrations 5,12,13 , we used both IVIg preparations at a final concentration of IgG of 10 mg/mL.We found that, as already reported for T cells from healthy donors, IVIgG impaired the up-regulation of the activation markers CD25, CD69 and PD-1, while IVIgGMA did not modify their expression (Fig. 1a-c).When the effects of both preparations were compared, we observed that the up-regulation of CD25 and CD69 was significantly lower in the presence of IVIg than in the presence of IVIgGMA (Fig. 1a,b), while no differences were found for PD-1 (Fig. 1c).The inhibition on T cell-activation mediated by IVIgG was dose-dependent showing to be statistically significant at 10 and 1 mg/mL but not at lower doses as previously described for T cells from healthy donors 5 (see Supplementary Fig. S1 online).Moreover, as shown in Fig. 1d, none of the IVIg preparations affected the viability of CD3 + cells of CLL patients. ", "section_name": "Results", "section_num": null }, { "section_content": "Then we evaluated the effect of the different IVIg preparations on T cell proliferation in response to TCR-stimulation and also in response to IL-15, a cytokine involved in homeostatic proliferation of memory T cells.To that aim CFSE-stained PBMC from CLL patients were cultured with immobilized anti-CD3 mAb or IL-15 in the presence of IVIgG, IVIgGMA or HSA.As shown in Fig. 2a, b we found that, contrary to what happened with early activation markers, both IVIg preparations were able to impair T cell proliferation when cells were stimulated through the TCR.This was observed both on CD4 + (Fig. 2a) and CD8 + T cells (Fig. 2b).Nevertheless, proliferation of T cells in response to TCR-stimulation was significantly lower in the presence of IVIgG than in the presence of IVIgGMA (Fig. 2a,b).T cell proliferation in response to IL-15 was impaired in CD4 + , but not in CD8 + T cells, only by IVIgG preparation (Fig. 2c,d).Again, the proliferation in response to IL-15 was lower in the presence of IVIgG than in the presence of IVIgGMA (Fig. 2c,d).Same results were found when the proliferation of CD8 + T cells in response to IL-2 was evaluated (see Supplementary Fig. S2 online). As shown in the Supplementary Fig. S3 online, the inhibitory effect of the IVIg preparations on the proliferation of T cells from CLL patients was due to a direct effect on this cell population, given that same results were obtained with purified T cells (> 95% T cells). ", "section_name": "The proliferation of T cells from CLL patients in response to TCR-stimulation or IL-15 is differentially affected by the two IVIg preparations.", "section_num": null }, { "section_content": "As mentioned before, it was recently reported that a SCIg preparation impaired CLL cell activation when stimulated through the BCR in vitro 10 .We asked if the IVIg preparations evaluated herein were also able to decrease leukemic B cell activation.To that aim, PBMC from CLL patients were activated with immobilized anti-IgM mAb to induce BCR crosslinking, in the presence of IVIgGMA, IVIgG or HSA, and after 24 h the expression of the activation markers CD69 and CD86 was assessed.As shown in Fig. 3a,b, both preparations decreased the upregulation of CD69 and CD86, without affecting CD19 + cell viability (Fig. 3c).In this case, the inhibitory effect of both preparations was not statistically significant different.The inhibitory effect did not depend on the presence of accessory cells given that similar results were observed with purified leukemic B cells (see Supplementary Fig. S4 online).Moreover, the inhibition on the up-regulation of the activation markers was accompanied with a decrease in the signaling pathway downstream the BCR as shown by a reduced phosphorylation of key molecules such as Syk, Btk and Erk 1/2 (Fig. 4). The up-regulation of the activation markers CD69 and CD86 in response to BCR cross-linking was significantly reduced by IVIgGMA at 1 and 10 mg/mL or IVIgG at 10 mg/mL, while both preparations at 0.1 mg/mL had no effect (see Supplementary Fig. S5 online).Moreover, the inhibition exerted by both preparations at 10 mg/ mL on CLL cell activation seems not to be a general effect but rather specific to particular signalling pathways, given that the inhibition was not observed on CXCL12 or CpG-activated CLL cells (see Supplementary Fig. S6 online). ", "section_name": "Both preparations of IVIg impaired B cell activation in response to BCR crosslinking.", "section_num": null }, { "section_content": "Although we observed that IVIg preparations did not affect the spontaneous apoptosis of leukemic cells (Fig. 1d,3c), we asked whether these preparations might affect the apoptosis induced by the BCL-2 inhibitor, venetoclax, currently employed in CLL treatment.To that aim PBMC from CLL patients were cultured with clinically relevant doses of venetoclax in the presence of IVIgGMA, IVIgG or HSA.We found that none of the IVIg preparations affect CLL cell apoptosis induced by venetoclax (Fig. 5a,b).Given that we have previously reported that venetoclax induces the apoptosis of T cells from CLL patients 14 , we also evaluated the effect of IVIg preparations on this cell population.Interestingly we found that IVIgGMA reduced T cell apoptosis induced by venetoclax while IVIgG did not (Fig. 5c,d). ", "section_name": "IVIgGMA reduced T cell, but not CLL cell, apoptosis induced by venetoclax.", "section_num": null }, { "section_content": "The potential benefit of IVIg preparations as an immune-modulator emerged when the infusion of high doses of IVIg in a patient with antibody deficiency and autoimmune thrombocytopenia results in the restoration of the platelets levels to its normal range 15 .Since that initial observation, the use of Igs preparations in the treatment of autoimmune and inflammatory conditions has significantly increased.The study of the mechanisms behind this immunomodulatory effect has expanded as well, and several, not mutually excluding mechanisms have been proposed, including the inhibition of T and B cell activation 4,16 .In CLL, the immunomodulatory capacity of Ig preparations was first suggested in a study from Besa, et al. when they observed a decrease in leukemic cell counts in CLL patients treated with IVIg for recurrent infections and/or autoimmune complications 17 .This observation was recently supported by the work of Spaner, et al. who showed an association between IgG levels above 8-9 mg/mL and a benign course of the disease 10 .They also reported that a SCIg preparation has inhibitory effects on leukemic cells in vitro. Here we show that two IVIg preparations, one with mainly IgG, and the other enriched with IgA and IgM, interfere with the BCR signalling pathway, decreasing the phosphorylation of early signalling molecules downstream the BCR, as Syk and Btk, and the activation of leukemic cells in response to BCR-stimulation in vitro.Considering the central role of BCR-signalling on CLL pathogenesis, the capacity to inhibit leukemic cell activation adds an attractive immunomodulatory effect to IVIg preparations for CLL patients.The inhibitory effect of both IVIg preparations on BCR-activated leukemic cells did not depend on the presence of accessory cells, since the inhibitory effect was observed either using PBMC or purified leukemic cells from CLL patients.Although we do not know the mechanism by which these preparations impair BCR signalling on leukemic cells, we do know that CLL cells express both FcɣRIIb and CD22 18 , which are receptors that negatively modulate BCR-mediated activation on B cells by interacting with IgG.In that regard, others have shown that blocking FcɣRIIb receptors on CLL cells abrogates IgG mediated inhibition of the activation through the BCR 10 .Also, sialylated IgG present in these preparations might bind CD22 and diminish BCR activation as reported for normal B cells 19 . We also found that IVIgG impaired the activation of T cells from CLL patients in response to TCR-stimulation, and also in response to other two soluble factors, IL-2 and IL-15, a cytokine involved in homeostatic proliferation of memory T cells.Interestingly, we found that the IVIgGMA preparation had no effect on the upregulation of early activation markers on TCR-stimulated T cells and on the proliferation in response to IL15 or IL-2, while it was able to decrease the proliferation in response to TCR-stimulation.Remarkably IVIgGMA showed a significantly lower inhibitory effect on T cells compared to the IVIgG preparation.The IVIgGMA Pentaglobin, differs from other IVIg preparations, not only in its particular isotype composition (76% IgG, 12% IgM and 12% IgA), but also in its manufacturing process.In order to induce virus inactivation, this IVIgGMA is treated with β-propiolactone, a treatment that also modifies amino-acid residues in the Fc domain of the IgG affecting its binding capacity to monocytes through FcɣR and also its complement fixation capacity 20 .Similar to what we have observed, others have reported that an anti-CMV hyperimmunoglobulin preparation treated with β-propiolactone was less effective in suppressing human T-cell activation in vitro compared to the same Ig preparation without this treatment.Thus, it is possible that β-propiolactone treatment is involved in the lower inhibitory capacity of the IVIgGMA preparation, although we cannot rule out the possibility that the different isotype composition also has consequences on its inhibitory capacity. When we evaluated the effect of IVIg preparations on the apoptosis induced by venetoclax, we observed that none of the two preparations affected CLL cell viability, while T cell apoptosis induced by venetoclax was significantly lower in the presence of IVIgGMA.Both, leukemic and T cells from CLL patients express high levels of the IgM receptor, the FcµR 21 .Although this receptor was originally described as anti-apoptotic in T cells 22 , convincing studies published later have demonstrated that the receptor has not an anti-apoptotic function per se 23 .Thus the mechanism behind this interesting observation deserves further study. Our study has two main limitations.First, the fact that both IVIg preparations have differences in their isotype composition along with differences in the production process (mainly β-propiolactone treatment), does not allow us to provide a conclusive explanation for their different effects.Second, patients with unmutated IGVH genes are underrepresented in our cohort (see Supplementary Table S1 online).Considering that this group of patients obtain particular benefit from venetoclax 24 , the observation that pentaglobin prevent T cell apoptosis induced by this drug should be validated with a larger cohort of unmutated patients.cells) were incubated with IVIgGMA, IVIgG (10 mg/mL of IgG) or HSA and anti-IgM (25 µg/mL) or the corresponding isotype control for 2 or 10 min.Then, cells were lysed and whole cell extracts were prepared as described in Material and Methods.Proteins were separated on a standard 10% SDS-PAGE and transferred to a PVDF membrane.Membranes were probed with primary antibodies for phospho-Syk (pSyk), phospho-Btk (pBtk), phospho-Erk1/2 (pErk) and β-actin, followed by the corresponding secondary antibody as described in Material and methods.Specific bands were visualized by enhanced chemiluminiscence (ECL) method.(a) Western blot analysis of pBtk, pSyk and pErk1/2 protein levels.β-actin was included as loading control.Cropped western blot images are shown, full-length blots are presented in Supplementary Fig. S7 CLL patient samples and cell separation procedures.The study was approved by the local ethics committee from Academia Nacional de Medicina, Buenos Aires, Argentina, according to the institutional guidelines (Approval number 15/20/CEIANM).Peripheral blood samples were obtained from CLL patients after signed informed consent.The study was conducted according to the principles of the Declaration of Helsinki. CLL was diagnosed according to standard clinical and laboratory criteria.At the time of the analysis patients were free from clinically relevant infectious complications and were either untreated or had not received treatment for a period of at least 6 months before investigation.Clinical characteristics of CLL patients included in the study are shown in Supplementary Table S1. Peripheral blood samples were obtained from CLL patients and peripheral blood mononuclear cells (PBMC) were isolated as previously described 14 . T cells from CLL patients were purified by positive selection with the anti-CD3 Microbead isolation kit (purity obtained > 95%).Leukemic B cells from CLL patients were obtained by negative selection with the anti-B-CLL Microbead isolation kit (purity obtained > 98%).Magnetic separation was performed according to manufacturer's instructions. ", "section_name": "Discussion", "section_num": null }, { "section_content": "For T cell activation, PBMC (3 × 10 5 cells/150 µL RPMI 10% FCS) were pre-incubated for 30 min at 37 °C with IVIgGMA, IVIgG (0.1-1-10 mg/mL of IgG) or HSA at equimolar concentration (control) and then cultured on a 96-well culture plate containing immobilized anti-CD3 mAbs (0.5 µg/mL) or the corresponding isotype control for 24 h at 37 °C.Then, cells were stained with mAb for CD4, CD8, CD25, CD69 and PD1 and evaluated by flow cytometry as detailed in the Flow cytometry section.The presence of HSA did not affect T cell activation (not shown). T cell proliferation was evaluated using the CFSE dilution assay.PBMC or purified T cells, both from CLL patients (3 × 10 5 cells/150 µL RPMI 10% FCS) were labelled with CFSE (1 µM) and then pre-treated for 30 min at 37 °C with IVIgGMA, IVIgG (10 mg/mL of IgG) or HSA.Then, cells were cultured on a 96-well culture plate containing immobilized anti-CD3 mAbs (0.5 µg/mL) or the corresponding isotype control or IL-15 (20 ng/ mL) or IL-2 (600 U/mL) for 5 days at 37 °C.Cells were then collected, stained with mAb for CD4 and CD8 and proliferation evaluated by flow cytometry.Percentage of proliferation was determinate as the % of T cells with low stain of CFSE. B cell cultures.PBMC or purified B-CLL cells (3 × 10 5 cells/150 µL RPMI 10% FCS) from CLL patients were pre-incubated with immobilized anti-IgM mAbs (25 µg/mL) or the corresponding isotype control for 30 min at 4 °C.Then, IVIgGMA, IVIgG (0.1-1-10 mg/mL of IgG) or HSA was added, and cells were cultured for 24 h at 37 °C.The expression of CD19, CD86 and CD69 was evaluated by flow cytometry.The presence of HSA did not affect B cell activation (not shown). PBMC were pre-treated for 30 min at 37 °C with IVIgGMA, IVIgG or HSA and then CpG (1 µM) or CXCL12 (500 ng/mL) were added to cultures.After 24 h, B cell activation was evaluated by flow cytometry. Venetoclax-induced apoptosis cultures.PBMC from CLL patients (3 × 10 5 cells/150 µL RPMI 10% FCS) were pre-treated in a 96-well plate for 30 min at 37 °C with IVIgGMA, IVIgG (10 mg/mL of IgG) or HSA. ", "section_name": "T cell cultures.", "section_num": null } ]	[ { "section_content": "The authors would like to thank to María Tejeda and Romina Mariel Pagano from CONICET for their technical assistance, and Guillermo Atenza from Microsules Argentina for the support on the project and helpful discussions. ", "section_name": "Acknowledgements", "section_num": null }, { "section_content": "This work was supported by grants and fellowships from the Agencia Nacional de Promoción Científica y Tecnológica (PICT 2017-2604), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Microsules Argentina. ", "section_name": "Funding", "section_num": null }, { "section_content": "www.nature.com/scientificreports/Then, different doses of Venetoclax (0.01-0.1-1 µM) or DMSO (drug vehicle) were added to cultures.After 24 h cell viability was assessed by flow cytometric alterations of light scattering properties and confirmed by staining with CD19, CD3 and Annexin V. Flow cytometry.For surface staining, cells were incubated with the corresponding antibodies, anti-CD3, CD4, CD5, CD8, CD19, CD25, CD69 and CD86, for 30 min at 4 °C.The staining was performed in phosphatebuffered saline-0.5%BSA.Cells were then washed and fixed with paraformaldehyde 1%.Gating of populations positive for any particular marker was based on fluorescence minus one (FMO) control of each activation marker.FMO was not modified by activation or IVIg treatment (data not shown). Cell viability was determined by Annexin V staining performed on binding buffer.After cell surface staining, cells were washed once with binding buffer and then stained with Annexin V for 20 min at room temperature.Samples were then acquired in the flow cytometry. Cell viability was also determined by flow cytometry by evaluating flow cytometric alteration of light scattering properties as previously described 14,27 .Briefly, apoptotic lymphocytes can be distinguished from viable lymphocytes by flow cytometry by their differences in cell morphology 28 .Apoptotic cells show a reduction in cell size (lower FSC), and as a result of chromatin condensation, nucleus fragmentation and cytoplasmic protein cross-linking, in the late stages of the apoptosis process the scattering of light in SSC is decreases (lower SSC) 29 .The analysis was performed evaluating the FSC-H vs SSC-H parameters, both on a linear scale. Samples were acquired with a BD FACSCalibur (BD Biosciences) and data were analyzed with FlowJo 10 software (FlowJo, USA). Western blot.Purified B-CLL cells (2 × 10 6 cells) were activated with anti-IgM (25 µg/mL) or the corresponding isotype control at 37 °C, in the presence of IVIgGMA, IVIgG (10 mg/mL of IgG) or HSA.After 2 or 10 min, the reaction was stopped with cold saline solution.Whole-cell lysates were prepared using 75 µL of RIPA buffer containing protease inhibitors (Thermo Fischer Scientific, #78440).Lysates were vortex and incubated on ice twice, and then after centrifugation, supernatants were transferred to a new tube.25 µL of loading buffer 4× containing β-mercaptoethanol was added and then samples were incubated for 5 min at 95 °C.50 µL of protein extracts were separated on a standard 10% SDS-PAGE and transferred to PVDF membranes (GE HealthCare Science, #GE1060023).Membranes were then blocked with a PBST solution containing 5% non-fat dry milk for 2 h at room temperature.Then, PVDF membranes were cut to perform the incubation of the different sections of the same membrane with different primary antibodies, as previously reported 30 .Membranes were incubated with primary antibodies over night at 4 °C as follows: membranes with molecular weight marker between 100 and 50 kDa were probed with anti-phospho-SYK and anti-phospho-BTK and membranes with molecular weight marker below 50 kDa were probed with anti-phospho-ERK1/2 and anti-β-actin.Membranes were then incubated with the corresponding secondary antibody, HRP-conjugated anti-rabbit or anti-mouse IgG mAb, for 1 h at room temperature.Specific bands were visualized by enhanced chemiluminiscence (ECL) method.The expression of β-actin was used as a loading control to normalize the protein levels detected in each lane of the same gel.The molecular weight marker was the Precision Plus Protein™ All Blue Prestained Protein Standards (10-250 kDa) from BioRad (#1610373).Densitometric measurements of specific bands were determinate by using ImageJ software (NIH). Statistical significance was determined using non-parametric tests: Wilcoxon matchedpairs signed rank test to compare between two paired groups and Friedman followed by the Dunn's post-test to compare three or more groups.Two-tailed tests were used and p < 0.05 was considered statistically significant.The corresponding p value is indicated.Data were analysed using the GraphPad Prism software version 7. The disclosures are the following: Bezares RF received compensation as speaker from Varifarma, Microsules, AstraZeneca and Abbvie.Borge M received a scientific research grant from Microsules and compensation as speaker from Bristol-Myers Squibb.The remaining authors declare no competing financial interests. The online version contains supplementary material available at https:// doi.org/ 10. 1038/ s41598-021-92412-8. Correspondence and requests for materials should be addressed to M.B. Reprints and permissions information is available at www.nature.com/reprints. ", "section_name": "Statistical analysis.", "section_num": null }, { "section_content": "www.nature.com/scientificreports/Then, different doses of Venetoclax (0.01-0.1-1 µM) or DMSO (drug vehicle) were added to cultures.After 24 h cell viability was assessed by flow cytometric alterations of light scattering properties and confirmed by staining with CD19, CD3 and Annexin V. Flow cytometry.For surface staining, cells were incubated with the corresponding antibodies, anti-CD3, CD4, CD5, CD8, CD19, CD25, CD69 and CD86, for 30 min at 4 °C.The staining was performed in phosphatebuffered saline-0.5%BSA.Cells were then washed and fixed with paraformaldehyde 1%.Gating of populations positive for any particular marker was based on fluorescence minus one (FMO) control of each activation marker.FMO was not modified by activation or IVIg treatment (data not shown). Cell viability was determined by Annexin V staining performed on binding buffer.After cell surface staining, cells were washed once with binding buffer and then stained with Annexin V for 20 min at room temperature.Samples were then acquired in the flow cytometry. Cell viability was also determined by flow cytometry by evaluating flow cytometric alteration of light scattering properties as previously described 14,27 .Briefly, apoptotic lymphocytes can be distinguished from viable lymphocytes by flow cytometry by their differences in cell morphology 28 .Apoptotic cells show a reduction in cell size (lower FSC), and as a result of chromatin condensation, nucleus fragmentation and cytoplasmic protein cross-linking, in the late stages of the apoptosis process the scattering of light in SSC is decreases (lower SSC) 29 .The analysis was performed evaluating the FSC-H vs SSC-H parameters, both on a linear scale. Samples were acquired with a BD FACSCalibur (BD Biosciences) and data were analyzed with FlowJo 10 software (FlowJo, USA). Western blot.Purified B-CLL cells (2 × 10 6 cells) were activated with anti-IgM (25 µg/mL) or the corresponding isotype control at 37 °C, in the presence of IVIgGMA, IVIgG (10 mg/mL of IgG) or HSA.After 2 or 10 min, the reaction was stopped with cold saline solution.Whole-cell lysates were prepared using 75 µL of RIPA buffer containing protease inhibitors (Thermo Fischer Scientific, #78440).Lysates were vortex and incubated on ice twice, and then after centrifugation, supernatants were transferred to a new tube.25 µL of loading buffer 4× containing β-mercaptoethanol was added and then samples were incubated for 5 min at 95 °C.50 µL of protein extracts were separated on a standard 10% SDS-PAGE and transferred to PVDF membranes (GE HealthCare Science, #GE1060023).Membranes were then blocked with a PBST solution containing 5% non-fat dry milk for 2 h at room temperature.Then, PVDF membranes were cut to perform the incubation of the different sections of the same membrane with different primary antibodies, as previously reported 30 .Membranes were incubated with primary antibodies over night at 4 °C as follows: membranes with molecular weight marker between 100 and 50 kDa were probed with anti-phospho-SYK and anti-phospho-BTK and membranes with molecular weight marker below 50 kDa were probed with anti-phospho-ERK1/2 and anti-β-actin.Membranes were then incubated with the corresponding secondary antibody, HRP-conjugated anti-rabbit or anti-mouse IgG mAb, for 1 h at room temperature.Specific bands were visualized by enhanced chemiluminiscence (ECL) method.The expression of β-actin was used as a loading control to normalize the protein levels detected in each lane of the same gel.The molecular weight marker was the Precision Plus Protein™ All Blue Prestained Protein Standards (10-250 kDa) from BioRad (#1610373).Densitometric measurements of specific bands were determinate by using ImageJ software (NIH). ", "section_name": "", "section_num": "" }, { "section_content": "Statistical significance was determined using non-parametric tests: Wilcoxon matchedpairs signed rank test to compare between two paired groups and Friedman followed by the Dunn's post-test to compare three or more groups.Two-tailed tests were used and p < 0.05 was considered statistically significant.The corresponding p value is indicated.Data were analysed using the GraphPad Prism software version 7. ", "section_name": "Statistical analysis.", "section_num": null }, { "section_content": "", "section_name": "Author contributions", "section_num": null }, { "section_content": "The disclosures are the following: Bezares RF received compensation as speaker from Varifarma, Microsules, AstraZeneca and Abbvie.Borge M received a scientific research grant from Microsules and compensation as speaker from Bristol-Myers Squibb.The remaining authors declare no competing financial interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "", "section_name": "Additional information", "section_num": null }, { "section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1038/ s41598-021-92412-8. Correspondence and requests for materials should be addressed to M.B. Reprints and permissions information is available at www.nature.com/reprints. ", "section_name": "Supplementary Information", "section_num": null } ]
10.1038/s41392-023-01337-3	PMN-MDSCs modulated by CCL20 from cancer cells promoted breast cancer cell stemness through CXCL2-CXCR2 pathway	<jats:title>Abstract</jats:title><jats:p>Our previous studies have showed that C-C motif chemokine ligand 20 (CCL20) advanced tumor progression and enhanced the chemoresistance of cancer cells by positively regulating breast cancer stem cell (BCSC) self-renewal. However, it is unclear whether CCL20 affects breast cancer progression by remodeling the tumor microenvironment (TME). Here, we observed that polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were remarkably enriched in TME of CCL20-overexpressing cancer cell orthotopic allograft tumors. Mechanistically, CCL20 activated the differentiation of granulocyte-monocyte progenitors (GMPs) via its receptor C-C motif chemokine receptor 6 (CCR6) leading to the PMN-MDSC expansion. PMN-MDSCs from CCL20-overexpressing cell orthotopic allograft tumors (CCL20-modulated PMN-MDSCs) secreted amounts of C-X-C motif chemokine ligand 2 (CXCL2) and increased ALDH<jats:sup>+</jats:sup> BCSCs via activating CXCR2/NOTCH1/HEY1 signaling pathway. Furthermore, C-X-C motif chemokine receptor 2 (CXCR2) antagonist SB225002 enhanced the docetaxel (DTX) effects on tumor growth by decreasing BCSCs in CCL20<jats:sup>high</jats:sup>-expressing tumors. These findings elucidated how CCL20 modulated the TME to promote cancer development, indicating a new therapeutic strategy by interfering with the interaction between PMN-MDSCs and BCSCs in breast cancer, especially in CCL20<jats:sup>high</jats:sup>-expressing breast cancer.</jats:p>	[ { "section_content": "In terms of cancer-related deaths among women, breast cancer is the most prevalent. 1,24][5] Recent studies indicated that the stemness of cancer cells was not exclusively regulated by intrinsic signals.4][15] However, the factors that influence the self-renewal of BCSCs remain ambiguous. Immature MDSCs with potent immunosuppressive activity accumulate during cancerous development. 16In mouse tumor model, two main MDSC subtypes have been characterized: monocytic myeloid-derived suppressor cells (M-MDSCs) labelled as CD11b + Ly6G -Ly6C high , and granulocytic polymorphonuclear MDSCs (PMN-MDSCs) labelled as CD11b + Ly6G + Ly6C low .M-MDSCs exhibit a stronger immunosuppressive effect compared to PMN-MDSCs. 16Nonetheless, PMN-MDSCs play an important role in the regulation of tumor featured immune responses. 17,18In cancer, MDSCs are significantly expanded and activated since they originate from myeloid progenitors. 19GMP differentiation into granulocyte progenitors (GPs) or monocyte progenitors (MPs) results in the production of PMN-MDSCs or M-MDSCs respectively. 20DSCs have been reported to promote the cancer progression through supporting cell survival, invasion and metastases, and angiogenesis. 16,17,21Recently, MDSCs were identified as a modulator of CSCs.MDSCs elevated microRNA-101 expression in ovarian cancer cells and promoted the CSC phenotype. 22MDSCs were also reported to endow multiple myeloma cell stemness by inducing piRNA-823 expression and DNA methyltransferase 3 beta (DNMT3B) activation. 23Peng et al found that MDSCs promoted breast cancer cell stemness through activating IL-6/STAT3 and NO/NOTCH cross-talk signaling. 13PMN-MDSC-derived exosomal S100 calcium-binding protein A9 (S100A9) was reported to promote the stemness in colorectal cancer. 24However, the regulation and mechanism of PMN-MDSCs on BCSCs were unclear. 6][27] Emerging evidence strongly suggested CCL20 was prominently upregulated in all breast cancer subtypes, especially basal-like subtypes. 28In agreement with previous reports, we recently identified that elevated level of CCL20 in blood serum and tumor tissues of breast cancer patients was linked to tumor malignancy and chemotherapy resistance. 28Some evidences showed that CCL20 might promote immunosuppressive TME to support tumor progression. 25,29For example, Tregs were induced pSIN and recruited to tumor tissues by CCL20 and increased colorectal cancer progression. 30,31High infiltration of CCR6 + Tregs suppressed the functions of IFNγ + CD8 + T cells to promote immunosuppression and disease progression in breast cancer. 32CL20 also facilitated the recruitment of T helper type 17 (Th17) cells through upregulating IL6/CCAAT/enhancer-binding protein β in cervical cancer progression. 33Additionally, CCL20 high expression increased DC infiltration into breast cancer tissues and promoted immunosuppression by reprograming DCs. 34These results drove us to further elucidate the mechanism that CCL20 regulated immunosuppression in breast cancer progression. Here, our study described that CCL20 promoted the differentiation of GMPs into GPs in the bone marrow (BM) by binding to its receptor CCR6, which resulted in a significant accumulation of PMN-MDSCs.CCL20-modulated PMN-MDSCs secreted amounts of CXCL2 and activated NOTCH1/HEY1 signaling pathway in breast cancer cells by binding to CXCR2, leading to the increase of ALDH + BCSCs.CXCR2 knockdown in breast cancer cells diminished the PMN-MDSC-induced enhancement of breast cancer cell stemness.Furthermore, CXCR2 antagonist SB225002 combined with DTX in vivo not only dramatically inhibited the tumor growth but also significantly decreased stemness of breast cancer cells, suggesting CXCR2 may be a potential therapeutic target for breast cancer patients with high expression of CCL20. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "To investigate whether CCL20 promoted cancer progression via remodeling TME, we established tumor-bearing Balb/c and C57BL/ 6N mouse models, which were orthotopically transplanted with pSIN-/CCL20-overexpressing 4T1 and Py8119 cells, respectively.CCL20 overexpression was confirmed at mRNA level by qRT-PCR and at the protein level in the blood serum of mice bearing CCL20-overexpressing breast cancer cell orthotopic allograft tumors by mouse CCL20 ELISA kit (Fig. 1a,b and Supplementary Fig. S1).We observed that CCL20 overexpression remarkably promoted tumor growth (Fig. 1c-e and Supplementary Fig. S2).Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database showed that the expression of CCL20 was positively correlated with the expression of CD11b (a myeloid marker in human, p value = 6.8e-25,R = 0.31) in patients' breast tumors (Supplementary Fig. S3). In our study, in line with a substantial expansion of MDSCs (Fig. 1f), a dramatic decrease of CD8 + T cells, especially CD8 + IFNγ + T cells (Fig. 1g,h), was observed in CCL20-overexpressing 4T1 tumors.No change was observed in macrophages, including inflammation-promoting M1-like as well as anti-inflammatory and immunosuppressive M2-like macrophages, DCs, and natural killer (NK) cells (Supplementary Fig. S4).Consistent with the results in tumor mouse models, the expression of CCL20 was positively correlated with the expression of CD33 (a MDSC marker in human, p value = 1.2e-15,R = 0.24) but not CD68 (a pan-macrophage marker in human, p value = 0.011, R = 0.077) in patients' breast tumors from GEPIA2 database (Supplementary Fig. S5), indicating that CCL20 overexpression facilitated cancer cells to create a tolerogenic environment. To further determine which MDSC subtype was increased in CCL20-overexpressing cell allograft tumors, the proportions of PMN-MDSCs and M-MDSCs were analyzed based on cell surface marker expression.Results of flow cytometry and immunofluorenscence (IF) consistently showed that the PMN-MDSC percentage was significantly increased in CCL20-overexpressing cell allograft tumors (Fig. 1i, j, l and Supplementary Fig. S6a).Consistently, the PMN-MDSC percentage in the spleen, blood, and BM was also elevated (Fig. 1i, j and Supplementary Figs.S6a,S7).In contrast, the M-MDSC percentage was decreased, indicating PMN-MDSCs were the main contributor to the MDSC expansion in CCL20overexpressing cell allograft tumors (Fig. 1k and Supplementary Fig. S6b).Furthermore, we examined the expressions of CD33 and CD15 in 31 breast tumor samples by immunohistochemistry (IHC) and found staining scores of these two PMN-MDSC markers were much higher in CCL20 high -expressing tumors than in CCL20 lowexpressing tumors (Fig. 1m).These results confirmed that CCL20 overexpression in breast cancer cells promoted PMN-MDSC expansion, which might suppress host immunity activity. ", "section_name": "PMN-MDSC expansion was positively correlated with high expression of CCL20 in breast cancer", "section_num": null }, { "section_content": "To investigate how CCL20 induced PMN-MDSC expansion in tumors, we first isolated BM cells from 4T1 allograft mouse model and then treated BM cells with recombination mouse CCL20 protein (rmCCL20).After three days, we observed that rmCCL20 significantly increased PMN-MDSC percentage in BM cells (Fig. 2a).Isolated BM cells were also cultured with conditioned medium of pSIN-/CCL20-overexpressing 4T1 cells.PMN-MDSC expansion was more pronounced in BM cells cultured with the conditioned medium of CCL20-overexpressing cells (Fig. 2b). Based on the results in vitro, we speculated that CCL20 might regulate BM progenitor cell differentiation, contributing to PMN-MDSC expansion.GMP is the point of bifurcation between monocytic and granulocytic differentiation. 20We found the percentage of GMPs and GMP-derived GPs were significantly increased whereas MPs were decreased in BM of mice bearing CCL20-overexpressing 4T1 cell orthotopic allograft tumors by flow cytometry (Fig. 2c and Supplementary Fig. S8a,b).The elevated GP/MP ratio reinforced the expansion of GPs (Fig. 2d and Supplementary Fig. S8c), revealing that CCL20 skewed GMP differentiation toward the granulocytic lineage, which gave rise to PMN-MDSCs. Fig. 1 PMN-MDSCs were significantly augmented in mice bearing CCL20-overexpressing breast cancer cell orthotopic allograft tumors.a pSIN-/CCL20-overexpressing 4T1 cell lines were established, and overexpression efficiency of CCL20 was confirmed by qRT-PCR.Bar graph was presented as the mean of three biological independent experiments (mean ± SEM).Balb/c mice were orthotopically transplanted with pSIN-/CCL20-overexpressing 4T1 cells (5 × 10 4 ) at the fourth mammary fat pads (n = 5 for each group).b The level of CCL20 in the blood serum of Balb/c mice was measured by ELISA and shown in bar graph as mean ± SEM. c Tumor size was monitored every 3 days, and tumor volume was calculated.d, e Tumor weight (d) and tumor image (e) was shown after the mice were sacrificed.f The percentage of MDSC (CD45 + CD11b + Gr-1 + ) in pSIN-/CCL20-overexpressing 4T1 allograft tumors was analyzed by flow cytometry and shown in bar graph as mean ± SEM. g, h The percentage of CD3 + CD8 + T cells (g) and the percentage of CD3 + CD8 + IFNγ + T cells (h) from CD45 + cells in pSIN-/CCL20overexpressing 4T1 cell allograft tumors were analyzed by flow cytometry and shown in bar graph as mean ± SEM. i-k The gating strategies for the analysis of PMN-MDSCs (CD45 + CD11b + Ly6C low Ly6G + ) and M-MDSCs (CD45 + CD11b + Ly6C high Ly6G -) by flow cytometry were shown (i).The percentage of PMN-MDSC (j) and M-MDSC (k) in tumor, spleen, blood, and BM of Balb/c mice bearing pSIN-/CCL20-overexpressing 4T1 cell allograft tumors were shown in bar graph as mean ± SEM. l The percentage of CD11b-positive cells and Ly6G-positive cells in Balb/c mice bearing pSIN-/CCL20-overexpressing 4T1 cell allograft tumors were analyzed by IF staining.Scale bar, 50 μm.m CD33 and CD15 expression in CCL20 high and CCL20 low tumor tissues from breast cancer patients were analyzed by IHC staining.Representative images and the graph of H Scores (mean ± SEM) were shown.Scale bar, 100 μm.ns, no significance; p < 0.05, p < 0.01, p < 0.001, *p < 0.0001 In methylcellulose cultures supplemented with hematopoietic cytokines, GMPs formed colony-forming units (CFUs) including granulocyte-monocyte colony (CFU-GM), granulocyte-colony (CFU-G) and monocyte-colony (CFU-M), but GPs only gave rise to CFU-G. 35To determine whether PMN-MDSC expansion was due to GMP differentiation by CCL20, we treated GMPs from BM of mice bearing 4T1 cell allograft tumors with rmCCL20 and observed that CFU-G, not CFU-M, was strikingly increased in the presence of rmCCL20 (Fig. 2e and Supplementary Fig. S8d,e).Consistently, GMPs sorted from BM of mice bearing CCL20-overexpressing 4T1 cell allograft tumors formed more CFU-G colonies (Fig. 2f and Supplementary Fig. S8f).We further collected the cells from the GMP-derived colonies and found that PMN-MDSC percentage in the colonies derived from GMPs treated with rmCCL20 or sorted from mice bearing CCL20-overexpressing 4T1 cell allograft tumors was significantly increased (Fig. 2g,h).Additionally, The CFU-G number and the PMN-MDSC percentage in CFU-G colonies derived from GPs treated with rmCCL20 or sorted from mice bearing CCL20-overexpressing 4T1 cell allograft tumors were not changed in comparison to controls (Supplementary Fig. S8g-j).These results confirmed that CCL20 induced the PMN-MDSC expansion via promoting the differentiation of GMPs to GPs. ) at the fourth mammary fat pads (n = 5 for each group).After mice were sacrificed, the percentage of bone marrow progenitor cells, including GMPs (Lin -c-Kit + Sca-1 -CD34 + FcγR +/high Ly6C -CD115 low ), GPs (Lin -c-Kit + Sca-1 -CD34 + FcγR +/ high Ly6C + CD115 low ) and MPs (Lin -c-Kit + Sca-1 -CD34 + FcγR +/high Ly6C + CD115 high ) were analyzed by flow cytometry (c), and the ratio of GP/MP was shown as mean ± SEM (d).e GMPs were sorted from BM of mice bearing 4T1 cell allograft tumors and treated with rmCCL20 (10 ng/ml).One thousand GMPs per well were cultured in the methylcellulose-based medium for CFU assay for 10 days.The representative images of CFU-G were shown (left), and the number of CFU-G (right) was counted in bar graph as mean ± SEM.Scale bar, 100 μm.f GMPs were sorted from BM of mice bearing pSIN-/CCL20-overexpressing 4T1 cell allograft tumors.One thousand GMPs per well were cultured in the methylcellulose-based medium for CFU assay for 10 days.The representative images of CFU-G were shown (left), and the number of CFU-G (right) was counted in bar graph as mean ± SEM.Scale bar, 100 μm.g, h The cells from the GMP-derived colonies were collected, and the PMN-MDSC percentage was analyzed by flow cytometry and shown in bar graph as mean ± SEM. i GMPs were sorted from BM of mice bearing 4T1 cell allograft tumors.After being transiently transfected with CCR6-siRNA, GMPs were treated with rmCCL20 (10 ng/ml) or PBS for 10 days, and then the PMN-MDSC percentage was analyzed by flow cytometry.Bar graph was presented as the mean of three biological independent experiments (mean ± SEM).ns, no significance; p < 0.05, p < 0.01, p < 0.001 To date, only CCR6 was reported as the CCL20 receptor. 36,37To verify whether CCL20 induced GMP differentiation through binding to CCR6, CCR6 was knocked down by siRNAs in GMPs (Supplementary Fig. S9a-d), and then CFU assay and flow cytometry were performed. The results showed that CCR6 knockdown suppressed CCL20-induced GMP differentiation (Fig. 2i and Supplementary Fig. S9e), suggesting that CCL20 promoted the differentiation of GMPs to GPs through CCR6.Breast cancer progression and resistance to therapy are believed to be primarily caused by BCSCs. 38,39We observed that, besides the expansion of PMN-MDSCs, ALDH + BCSCs, not CD24 + CD29 + BCSCs, 5 were increased significantly in CCL20-overexpressing tumors (Fig. 3a, b and Supplementary Fig. S10).To determine whether CCL20-modulated PMN-MDSCs played a critical role in promoting the stemness of breast cancer cells, pSIN-or CCL20modulated PMN-MDSCs sorted from pSIN-/CCL20-overexpressing cell allograft tumors were co-cultured or mix-cultured with breast cancer cells for three days and the percentage of ALDH + BCSCs was analyzed by flow cytometry.Results showed that CCL20modulated PMN-MDSCs markedly elevated the percentage of ALDH + BCSCs (Fig. 3c, d and Supplementary Fig. S11a).However, there was no change for the percentage of ALDH + BCSCs in 4T1 cells co-cultured with M-MDSCs sorted from CCL20overexpressing cell allograft tumors (Supplementary Fig. S11b).These results indicated that CCL20-modulated PMN-MDSCs induced the enrichment of ALDH + BCSCs.In addition, the expressions of four stemness-related genes including Klf4, Nanog, Sox9 and Aldh1a1 were upregulated in 4T1 or Py8119 cells cocultured with CCL20-modulated PMN-MDSCs (Fig. 3e,f).Furthermore, we assessed the in vitro self-renewal ability of 4T1 cells mixcultured with pSIN-or CCL20-modulated PMN-MDSCs.As shown in Fig. 3g to i, CCL20-modulated PMN-MDSCs obviously promoted tumor sphere formation, and the mRNA expression levels of four stemness-related genes in primary mammosphere were remarkably increased (Supplementary Fig. S11c). Next, the limited dilution assay (LDA) was used to investigate the effect of CCL20-modulated PMN-MDSCs on BCSC self-renewal in vivo.At first, 100 and 500 4T1 cells co-cultured with pSIN-or CCL20-modulated PMN-MDSCs were injected in the mammary fat pads of female mice.Then, tumor volume was monitored for one month, The results of in vivo LDA demonstrated that the frequency of tumor-initiating cells was increased approximately six-fold in the group of 4T1 cells co-cultured with CCL20modulated PMN-MDSCs in comparison to the pSIN group (Fig. 3j,k).Collectively, these results indicated that CCL20modulated PMN-MDSCs enhanced the stemness and selfrenewal ability of breast cancer cells.CCL20-modulated PMN-MDSCs enhanced breast cancer cell stemness through the CXCL2-CXCR2 pathway To explore the mechanism that CCL20-modulated PMN-MDSCs promoted stemness of breast cancer cells, we sorted tumor cells and PMN-MDSCs from pSIN-/CCL20-overexpressing 4T1 cell allograft tumors and examined the transcriptome profiling via RNA-Seq.KEGG pathway gene set enrichment analysis (GSEA) revealed that chemokine signaling pathways were enriched in both tumor cells and PMN-MDSCs (Fig. 4a,b).We screened top ten upregulated genes in chemokine signaling pathways and the top fifty up-regulated genes in all sequenced genes, and consistently found CXCL2 and its receptor CXCR2 were significantly upregulated in CCL20-modulated PMN-MDSCs and tumor cells, respectively (Fig. 4c,d and Supplementary Fig. S12a,b).The upregulated mRNA levels of CXCL2 and CXCR2 were further confirmed by qRT-PCR (Fig. 4e,f).In addition, more CXCL2 was present in the conditioned medium of CCL20-modulated PMN-MDSCs compared to pSIN group as measured by mouse CXCL2 ELISA kit (Fig. 4g). To verify whether CCL20-modulated PMN-MDSCs promoted stemness of breast cancer cells via CXCL2-CXCR2 axis, 4T1 or Py8119 cells were treated with recombination mouse CXCL2 protein (rmCXCL2) for three days.The results of flow cytometry (Fig. 4h and Supplementary Fig. S13a), qPCR (Supplementary Fig. S13b,c), in vitro LDA (Fig. 4i and Supplementary Fig. S13d), and soft agar anchorage-independent growth (AIG) assay (Fig. 4j) consistently showed that rmCXCL2 increased the stemness properties of breast cancer cells.The increase of ALDH + BCSC percentage in 4T1 or Py8119 cells co-cultured with CCL20modulated PMN-MDSCs was blocked by CXCL2 neutralizing antibody (Fig. 4k and Supplementary Fig. S14a).Furthermore, utilizing qRT-PCR (Supplementary Fig. S14b,c), in vitro LDA (Supplementary Fig. S14d,e), and AIG assay (Supplementary Fig. S14f), we also confirmed that CXCL2 neutralizing antibody inhibited breast cancer cell stemness promoted by CCL20modulated PMN-MDSCs, suggesting CCL20-modulated PMN-MDSCs promoted the cancer cell stemness by secreting amounts of CXCL2. We sequentially evaluated the protein expression of CXCR2 in ALDH + BCSCs and found that CXCR2 protein was highly expressed in ALDH + BCSCs by western blotting (Supplementary Fig. S15), suggesting that CXCR2 might play a critical role in activating the stemness of breast cancer.Thus, we treated scramble or CXCR2knockdown 4T1 cell lines (Supplementary Fig. S16) with rmCXCL2 for three days.CXCR2 knockdown significantly inhibited the increase of ALDH + BCSCs induced by rmCXCL2 (Fig. 4l).Similar results were obtained using a co-culture system (Fig. 4m).In addition, mammosphere formation ability and stem-related gene expression were consistently suppressed significantly in CXCR2knockdown cells (Supplementary Fig. S17).These data indicated that CCL20-modulated PMN-MDSCs enhanced the stemness of BCSCs through the CXCL2-CXCR2 pathway. The CXCL2-CXCR2 axis enhanced the stemness of breast cancer cells relying on NOTCH1/HEY1 To reveal the signaling pathway involved in CXCL2-promoted breast cancer cell stemness, we treated 4T1 cells with rmCXCL2 or Fig. 3 CCL20-modulated PMN-MDSCs enhanced the stemness of breast cancer cells.Balb/c mice or C57BL/6N mice were orthotopically transplanted with pSIN-/CCL20-overexpressing 4T1 or Py8119 cells at the fourth mammary fat pads, respectively.a, b The percentage of ALDH + BCSCs was determined by ALDEFLUOR assay in tumor cells (CD45 -CD140b -CD31 -) from tumors of Balb/c mice (a) or C57BL/6N mice (b).Bar graph was presented as the mean of three biologically independent experiments (mean ± SEM).c, d PMN-MDSCs, sorted from pSIN-/ CCL20-overexpressing 4T1 or Py8119 cell allograft tumors, were labeled as pSIN PMN or CCL20 PMN in Figures and as pSIN-PMN-MDSCs or CCL20modulated PMN-MDSCs in main text.4T1 or Py8119 cells were co-cultured with pSIN-/CCL20-modulated PMN-MDSCs for 3 days.The percentage of ALDH + BCSCs was determined by ALDEFLUOR assay in 4T1 (c) or Py8119 cells (d).Bar graph was presented as the mean of three biologically independent experiments (mean ± SEM).e, f 4T1 or Py8119 cells were co-cultured with pSIN-/CCL20-modulated PMN-MDSCs for 3 days.The mRNA expression levels of several stemness-related genes were analyzed in 4T1 (e) or Py8119 cells (f) by qRT-PCR.Bar graph was presented as the mean of three biologically independent experiments (mean ± SEM).g-i Self-renewal capability was determined by forming both primary mammospheres (g, n = 7 for each group) and secondary mammospheres (h, n = 4 for each group) in 4T1 cells mix-cultured with pSIN-/CCL20-modulated PMN-MDSCs.The average cell number of each secondary mammosphere was shown (i), and bar graph was presented as mean ± SEM.Scale bar, 1 mm.j, k 4T1 cells were mix-cultured with pSIN-/CCL20-modulated PMN-MDSCs for 3 days, and then sorted by flow cytometry and engrafted to the fourth mammary fat pads of Balb/c mice at a limited dilution (n = 3 for each group, two injection sites each mouse, 100 or 500 cells/site).Tumor image was shown (j).The stem cell frequency in tumor tissue was calculated by the limited dilution assay (k).The stem cell frequency and p-value calculation were based on the positive tumor sites.p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 PBS and performed RNA-Seq analysis.Bioinformatic analysis revealed that CXCL2 activated the NOTCH signaling pathway, especially NOTCH1 signaling pathway (Fig. 5a and Supplementary Fig. S18).Previous studies have shown that NOTCH signaling is important for cancer progression and BCSC regulation. 40,41Hairy and enhancer of split (HES) as well as hes related family bHLH transcription factor with YRPW motif (HEY) families are both normally considered primary downstream targets of NOTCH signaling and contribute to cell fate determination. 42Therefore, the protein expressions of NOTCH1/2/3 and their intracellular domains NICD1/2/3 were determined by western blotting, and mRNA levels of downstream genes in HES and HEY families were analyzed by qRT-PCR.The results showed that NICD1 protein expression level and HEY1 mRNA level were remarkably upregulated (Fig. 5b,c and Supplementary Fig. S19a).However, the protein expressions of NICD1, HEY1, and Aldehyde dehydrogenase 1A1 (ALDH1A1) were inhibited significantly in CXCR2knockdown 4T1 cell lines (Fig. 5d and Supplementary Fig. S19b).Consistent results were obtained using the co-culture system (Fig. 5e, f and Supplementary Fig. S19c,d).These results indicated that NOTCH1/HEY1 signaling pathway was involved in CXCL2-CXCR2 axis-induced enrichment of BCSCs. To validate the role of NOTCH1/HEY1 pathway on mediating CXCL2-induced breast cancer cell stemness, 4T1 cells were treated with rmCXCL2 and NOTCH inhibitor RO4929097, a γ-secretase inhibitor to reduce NICD1 by blocking transmembrane proteolytic cleavage. 43The increase of ALDH + BCSCs induced by rmCXCL2 was inhibited in 4T1 cells under RO4929097 treatment (Fig. 5g).The elevated expressions of NICD1, HEY1, ALDH1A1 by rmCXCL2 were also decreased in 4T1 cells after RO4929097 treatment (Fig. 5h and Supplementary Fig. S19e).Consistent with the results, RO4929097 treatment strikingly inhibited ALDH + BCSCs and the protein expressions of NICD1, HEY1, ALDH1A1 in 4T1 cells cocultured with CCL20-modulated PMN-MDSCs (Fig. 5i,j and Supplementary Fig. S19f).Consistent results were observed in Py8119 cells (Supplementary Figs.S20,S21).Taken together, we confirmed that CXCL2-CXCR2 axis, which was activated by CCL20modulated PMN-MDSCs, enhanced the stemness of breast cancer cells by NOTCH1/HEY1 signaling pathway. CXCR2 antagonist enhanced the therapeutic efficacy of DTX on CCL20-expressing breast tumors Given the critical role and clinical relevance of CCL20-modulated PMN-MDSCs on promoting stemness of breast cancer cells, we sought to evaluate whether targeting CXCL2-CXCR2 axis could be an effective strategy to treat CCL20 high -expressing breast cancer patients who are prone to show poor response to DTX.In addition to the syngeneic mouse model, humanized hematopoietic stem cell-NOG-EXL (huHSC-NOG-EXL) mice, which express human interleukin-3 (hIL-3) as well as granulocyte-macrophage colonystimulating factor (GM-CSF) and show in general a superior humanization phenotype, 44,45 was constructed to elucidate the potential synergies of DTX and CXCR2 inhibition.We orthotopically transplanted pSIN-/CCL20-overexpressing 4T1 or Py8119 cells into the mammary fat pads of Balb/c or C57BL/6N mice, as well as MDA-MB-231 cells, which showed higher CCL20 expression (Supplementary Fig. S22), into the mammary fat pads of huHSC-NOG-EXL mice.We designed a novel treatment strategy by combining DTX with a potent and selective CXCR2 antagonist SB225002 in these orthotopic breast tumor mouse models 46,47 (Supplementary Fig. S23a,b) and found that the combination of DTX and SB225002 showed a more significant inhibiting effect on tumor growth than either treatment alone in both syngeneic mice and humanized mice bearing CCL20 high -expressing breast cancer cell allograft or xenograft tumors (Fig. 6a-d and Supplementary Figs.S23c, S24a-c).SB225002 treatment effectively decreased ALDH + BCSCs (Fig. 6e and Supplementary Figs.S24d,S25).Consistently, the combinational treatment showed the inhibition on synergistic effects on tumor shrinkage (Supplementary Fig. S26) and secondary tumor initiation (Fig. 6f and Supplementary Fig. S24e). These results showed that the CXCR2 antagonist obviously enhanced the therapeutic efficacy of DTX and might be a promising candidate to increase anticancer efficacy of DTX in breast cancer, especially in CCL20 high -expressing breast cancer, suggesting the combinational treatment may be more effective in CCL20 high -expressing breast cancer patients. ", "section_name": "CCL20 promoted PMN-MDSC expansion by inducing GMP differentiation to GPs", "section_num": null }, { "section_content": "High CCL20 expression levels were positively correlated to tumor stage and grade, as well as the occurrence of pleural metastases in human triple-negative breast cancer cell lines. 25,48Moreover, previous study showed that CCL20 could promote BCSC selfrenewal through the activation of nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) activity. 28evertheless, there is no evidence of a causative or functional link between TME regulated by CCL20 and breast cancer progression, particularly in BCSCs.In this study, we discovered for the first time that CCL20 modulated the PMN-MDSC expansion in BM, blood, spleen and tumor, promoting tumor immunesuppressive microenvironment.Mechanistically, CCL20 promoted the GMP differentiation to GPs via binding to its receptor CCR6, contributing to the PMN-MDSC expansion.GMPs have been found to be increased across a variety of tumor types in previous studies. 20Poorer clinical outcomes were associated with higher circulating GMPs, as reflected by clinical stages and reduced time to progression. 20Consistent with this process is our observation that GMPs in CCL20-overexpressing cell allograft mouse model were remarkably increased.It has been reported that lineageinstructive transcription factors, such as IFN regulatory factor 8 (IRF8), regulated the production and differentiation of GMPs. 35,49owever, the key molecules that influence GMP differentiation are poorly studied in cancer progression.In this study, we identified that the differential direction of GMPs was influenced by CCL20.CCL20 stimulated the GMP differentiation to GPs as resulting in the PMN-MDSC expansion in BM.These studies confirmed CCL20 as an important positive regulator of PMN-MDSC production.In Fig. 4 CXCL2 secreted by CCL20-modulated PMN-MDSCs enhanced the stemness through CXCR2.a-d Tumor cells and PMN-MDSCs were sorted from pSIN-/CCL20-overexpressing 4T1 cell allograft tumors.The total RNA of pSIN-/CCL20-modulated PMN-MDSCs (pSIN PMN /CCL20 PMN ) or tumor cells were extracted and used for RNA-Seq analysis.Bubble charts of gene function annotation and enrichment from tumor cells (a) or PMN-MDSCs (b) in pSIN-/CCL20-overexpressing 4T1 cell allograft tumors were shown.Significantly upregulated chemokines receptors in tumor cells (c) or chemokines from PMN-MDSCs (d) were screened and shown as heatmaps.The order was rearranged by variation based on the fold change.e, f qRT-PCR was performed to determine the mRNA expression levels of CXCL2 in pSIN PMN or CCL20 PMN (e) and CXCR2 in tumor cells (f) sorted from pSIN-/CCL20-overexpressing 4T1 cell allograft tumors.Data were presented as mean ± SEM. g pSIN PMN or CCL20 PMN from pSIN-/CCL20-overexpressing 4T1 or Py8119 cell allograft tumors were cultured for 2 days.The level of CXCL2 in the culture medium was measured and normalized by ELISA.Bar graph was shown as mean ± SEM. h 4T1 cells were treated with rmCXCL2 (10 ng/ml) for 3 days, and the percentage of ALDH + BCSCs was determined by ALDEFLUOR assay in 4T1 cells.Bar graph was presented as the mean of three biologically independent experiments (mean ± SEM).i In vitro LDA of 4T1 cells treated with rmCXCL2 (10 ng/ml) for 3 days.j 4T1 cells were treated with rmCXCL2 (10 ng/ml) for 3 days, and then the soft agar colony formation assay was performed.After 4-6 weeks, the colony images were taken (left), and the colony numbers were quantified (right).Bar graph was presented as the mean of three biologically independent experiments (mean ± SEM).k 4T1 cells were co-cultured with pSIN PMN or CCL20 PMN from 4T1 cell allograft tumors.Then, the CXCL2 neutralizing antibody (anti-CXCL2, 2 µg/ml) or control (anti-IgG, 2 µg/ml) was administrated for 3 days, and the percentage of ALDH + BCSCs was determined by ALDEFLUOR assay in 4T1 cells.Bar graph was presented as the mean of three biologically independent experiments (mean ± SEM).l Cells were treated with rmCXCL2 (10 ng/ml) or PBS for 3 days.The percentage of ALDH + BCSCs was determined by ALDEFLUOR assay in 4T1 cells, and bar graph was presented as mean ± SEM. m Cells were co-cultured with pSIN PMN or CCL20 PMN sorted from 4T1 cell allograft tumors for 3 days.the percentage of ALDH + BCSCs was determined by ALDEFLUOR assay in 4T1 cells, and bar graph was presented as the mean of three biologically independent experiments (mean ± SEM).ns, no significance; p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 future, concrete mechanisms on how CCL20-CCR6 axis influenced the differentiation of GMPs to GPs need to be elucidated. In the interactions between tumor cells and immune cells, the TME is the main battleground.In recent years, a great deal of research has focused on how the immune system modulates CSCs. 50Deregulated differentiation of myeloid-derived cells was reported to alter tumor phenotype by changing the plasticity of BCSCs. 51,52There is considerable evidence that PMN-MDSCs promote cancer cell stemness via numerous ways, including S100A9-positive exosome, piRNA-823 expression, DNMT3B activation, and IL-6/STAT3 and NO/NOTCH cross-talk signaling. 13,23,24In this study, we have yielded significant original insights into specific immunobiology and pathology between CCL20modulated PMN-MDSCs and CSCs in the context of breast cancer.progression, although further studies are required to clarify whether there is a specific subtype of PMN-MDSCs modulated by CCL20 to drive this loop.We investigated the key mediator in CCL20-modulated PMN-MDSCs that conveyed breast cancer cell stemness.Bioinformatic analysis showed that CXCL2 was positively related to CCL20 in breast cancer patients (p value = 8.4e-31, R = 0.34) (Supplementary Fig. S27), and CXCL2 was abundant and highly secreted by PMN-MDSCs in CCL20-overexpressing tumors.Considerable evidence showed that CXCL2 overexpression was poor prognosis in patients with ovarian cancer, cervical cancer, etc. 53,54 The upregulated CXCL2 expression in cancer cells was proposed to promote invasion and migration. 55Here, we demonstrated CXCL2 secreted by PMN-MDSCs in TME cooperated with its receptor CXCR2 which was highly expressed on BCSCs, indicating a cross-talk between PMN-MDSCs and BCSCs was achieved by CXCL2-CXCR2 axis.In addition, we observed the phenomenon that CXCL2 increased CXCR2 expression in vitro and in vivo.In addition, CXCR2 expression was higher in ALDH + BCSCs compared to ALDH -BCSCs (Supplementary Fig. S15).We speculated that the enrichment of ALDH + BCSCs induced by CXCL2 might contribute to the increased CXCR2 expression, which provided a positive feedback loop to promote the stemness of breast cancer cells.Specific mechanisms deserve to be further explored. DTX is one of the most widely used antimitotic chemotherapy drugs for the treatment of cancers. 56It has been reported that DTX possesses chemo-immunomodulatory properties besides its canonical antitumor properties. 57,58Our recent reports showed that DTX induced C-C motif chemokine ligand 3 (CCL3) secretion in macrophages and triggered proinflammatory polarization of macrophages to inhibit breast cancer progression. 59However, most patients eventually become chemo-resistant accompanied with tumor progression.There is considerable evidence that CSC enrichment played a key role in contributing to chemotherapy resistance of DTX. 28,602][63] In our study, we used SB225002, a selective CXCR2 antagonist with promising therapeutic effect in cancers, 46 to blocking CXCR2 in breast cancer cells.Our results demonstrated that CXCR2 blockade sensitized cancer cells to DTX via decreasing cancer cell stemness.Additionally, due to the increasing accumulation of PMN-MDSCs and enrichment of ALDH + BCSCs, the combination of DTX and CXCR2 antagonist had a better therapeutic effect in CCL20-overexpressing cell allograft mouse model.Collectively, these results indicated that CXCR2 blockade augmented chemotherapeutic effects of DTX, especially in CCL20 high -expressing breast cancer patients. In conclusion, we discovered that the CCL20 overexpression in breast cancer cells significantly promoted the PMN-MDSC expansion.The CCL20-modulated PMN-MDSCs in TME played indispensable roles in promoting breast cancer cell stemness.CXCR2 antagonist SB225002 combined with DTX may be a hopeful therapeutic strategy to overcome chemoresistance and gain better clinical outcomes in patients with breast cancer, especially CCL20 high -expressing breast cancer (Fig. 6g). ", "section_name": "DISCUSSION", "section_num": null }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "The Shanghai Cancer Center in Fudan University provided the tumor tissues for all the breast cancer patients.For each patient involved, informed consent was obtained.The study was approved by Fudan University Shanghai Cancer Center Institutional Review Board (050432-4-1212B). The animal experiments were strictly carried out in accordance with the People's Republic of China Legislation Regarding the Use and Care of Laboratory Animals and approved by the Fudan University Shanghai Cancer Center Institutional Review Board (JS-082). ", "section_name": "Ethics statements", "section_num": null }, { "section_content": "The breast cancer cell lines MDA-MB-231, 4T1, and Py8119 were purchased from ATCC.The culture medium for MDA-MB-231 and 4T1 was RPMI 1640 medium (Gibco) supplemented with 5% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Beyotime Biotechnology).The culture medium for Py8119 cells was F12 medium (Gibco) with 5% FBS (Gibco), 1 µg/ml hydrocortisone (Sigma), 50 µg/ml gentamicin (Sigma), 10 ng/ml epidermal growth factor (PeproTech) and 5 µg/ml insulin (Biosharp Life Science).HEK293T cells were cultured in DMEM (Gibco) medium supplemented with 10% FBS and 1% penicillin/streptomycin.All cell lines were evaluated and authenticated.These cell lines were maintained at 37 °C in a humidified atmosphere of 5% CO 2 incubator. Plasmid/short hairpin RNA (shRNA) construction and lentivirus transfection CCL20 was amplified using the complementary DNA (cDNA) of 4T1 cell line and cloned into pSIN-puro lentiviral vector (Addgene), and the authenticity was verified by sequencing.The shRNA sequence of CXCR2 from Sigma-Aldrich was cloned into pLKO.1purolentiviral vector (Addgene).Then, HEK293T cells were transfected with plasmid DNA to generate high titer lentivirus which was used to infect breast cancer cells to establish stable cell lines.The primers used for plasmid construction were listed in Supplementary Table 1. Mice and tumor models Female Balb/c mice, C57BL/6N mice as well as the huHSC-NOG-EXL were purchased from Charles River and housed in specific pathogen-free facilities at the Department of Laboratory Animal Science of Fudan University.For the determination of tumorigenicity, 4T1 cells (5 × 10 4 ), Py8119 cells (1 × 10 5 ) or MDA-MB-231 cells (2 × 10 6 ) were orthotopically injected into the fourth Fig. 6 CXCR2 antagonist enhanced the therapeutic efficacy of docetaxel on breast tumors.a, b Balb/c mice were orthotopically transplanted with pSIN-/CCL20-overexpressing 4T1 cells (2×10 4 ) at the fourth mammary fat pads for combinational treatment (n = 5 for each group).Tumor size was monitored every 3 days, and tumor volume was calculated (a).The tumor was weighed after mice were sacrificed (b).c, d huHSC-NOG-EXL mice were orthotopically transplanted with MDA-MB-231 cells (2 × 10 6 ) at the fourth mammary fat pads (n = 4 for each group).Tumor size was monitored every 3 days, and tumor volume was calculated (c).The tumor was weighed after mice were sacrificed (d).e The percentage of ALDH + BCSCs was determined by ALDEFLUOR assay in tumor cells from pSIN-/CCL20-overexpressing 4T1 cell allograft tumors and shown in bar graph as mean ± SEM. f Tumor cells (CD45 -CD140b -CD31 -) isolated from SB225002-and/or DTX-treated pSIN or CCL20overexpressing tumors were engrafted to mammary fat pads of Balb/c mice at a limited dilution (n = 3 for each group, two sites each mouse, 100 or 500 cells/site).The stem cell frequency and p-value calculation were based on the positive tumor sites.g The schematic diagram for the findings of the current studies.CCL20 promoted GMP differentiation to GPs in BM by binding to CCR6, resulting in the PMN-MDSC expansion.CCL20-modulated PMN-MDSCs secreted amounts of CXCL2 and then activated NOTCH1/HEY1 signaling pathway via binding to CXCR2 to enhance the stemness of breast cancer cells.CXCR2 antagonist SB225002 combined with DTX in vivo not only dramatically inhibited the tumor growth but also significantly decreased the stemness of breast cancer cells.ns, no significance; p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 mammary fat pads of Balb/c mice, C57BL/6N mice or huHSC-NOG-EXL mice, respectively.Tumor size was measured every 3 days and calculated as tumor volume = Length × Width 2 /2. CXCR2 antagonist SB225002 (Selleckchem) was suspended in special solvent that consisted of 30% polyethylene glycol, 5% Tween-80, 2% dimethyl sulfoxide, and 63% water.For the combination therapy of SB225002 and docetaxel (DTX) (Hengrui) in vivo, mice were randomly separated into the indicated groups when the average diameter of tumors reached approximately 2-3 mm.SB225002 (10 mg/kg, once every 3 days) or equal volume vehicle alone or combined with DTX (20 mg/kg for 4T1, 40 mg/kg for Py8119, once every 6 days) were administered by intraperitoneal injection. RNA isolation and quantitative real-time PCR Total RNA from samples was extracted with TRIzol reagent (Takara), and the cDNA was obtained by reverse transcription using the HiScript II 1st Strand cDNA Synthesis kit (Vazyme Biotech) and T100 Thermal Cycler (BIO-RAD).The quantitative realtime PCR (qRT-PCR) was performed with AceQ Universal SYBR qPCR Master Mix (Vazyme Biotech) using 7300Plus Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions.The mRNA level of each gene was expressed relative to reference gene.Relative expression value was calculated by using the comparative C t method (2 -ΔCt ).The primers used for qRT-PCR were listed in Supplementary Table 2. ", "section_name": "Cell culture", "section_num": null }, { "section_content": "The conditioned medium of established breast cancer cell lines and the blood serum of mice bearing breast cancer cell orthotopic allograft tumors were collected to determine CCL20 levels according to the manufacturer's instructions (RayBiotech).For the detection of CXCL2, PMN-MDSCs sorted from tumors were cultured for 2 days in vitro.The CXCL2 levels in the conditioned medium of PMN-MDSCs were determined with a Mouse CXCL2 ELISA Assay kit (RayBiotech) according to the manufacturer's instructions. Western blotting Cells were lysed with RIPA buffer (Beyotime Biotechnology) containing phenylmethylsulfonyl fluoride (PMSF) (Beyotime Biotechnology) on ice for 30 min.The protein lysates were quantified with a BCA kit (Thermo Fisher) and then denatured in loading buffer.Equal amounts of lysates were electrophoretically separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore).5% de-fat milk was used for blocking the membranes at room temperature (RT) for 1 h.Then, they were incubated with primary antibody at 4 °C overnight, followed by the incubation with HRP-conjugated secondary antibody at RT for 1 h.Chemiluminescence was detected to use an ImageQuant LAS 4000 Micro Imaging System (GE) with western HRP Substrate (Millipore).Intensities in the resulting bands were quantified by ImageJ software.The antibody information used for western blotting was shown in Supplementary Table 3. ", "section_name": "ELISA", "section_num": null }, { "section_content": "Single-cell suspensions were made from tumor tissues and different organs of mice bearing breast cancer cell orthotopic allograft tumors.For analysis of tumor and spleen, single-cell suspensions were prepared as previously described. 28For BM analysis, hind limb bones of mice (femurs and tibias) were cut off.After removing the fur and muscles, bones were flushed using a 1 ml sterile syringe, and then the obtained cells were passed through a 70 μm filter.For blood analysis, blood samples were collected in EDTA-coated tubes and centrifuged at 1200 rpm for 10 min to separate serum and plasma.The erythrocytes in plasma were lysed by ACK lysis buffer (BioLegend). When analyzing cancer cells, PE-conjugated anti-mouse CD45, CD31 and CD140b antibodies were used.For immune cell analysis, single-cell suspensions were firstly incubated with anti-mouse CD16/32 antibody to block the non-specific binding.To analyze the percentage of total MDSCs with its two subtypes PMN-MDSCs and M-MDSCs, the cells were labeled with fluorescenceconjugated antibodies to CD45, CD11b, Gr-1, Ly6C and Ly6G.To identify the subtypes of T cells, the cells were stained with CD45, CD3, CD8, followed by the intracellular staining of IFNγ.Macrophages were analyzed using antibodies to CD45, CD11b, F4/80, MHCII and CD206.For the identification of bone marrow progenitor cells, bone marrow cells were labeled with lineage marker (CD3, CD4, CD8a, CD19, CD45R/B220, CD127) and fluorescence-conjugated antibodies to c-Kit, Sca-1, CD34, FcγR, Ly6C and CD115.The viability of cells was analyzed by DAPI (Sigma) staining.The antibody information used for flow cytometries was shown in Supplementary Table 3. For ALDEFLUOR assay (StemCell Technologies), dissociated single cells were resuspended in ALDEFLUOR buffer containing ALDEFLUOR substrate BAAA and incubated at 37 °C for 40 min.DEAB was added as a negative control.All the flow cytometry and cell sorting experiments were performed using MoFlo Astrios or CytoFlex instrument (Beckman Coulter) and analyzed by Summit 6.3 software. ", "section_name": "Isolation of breast cancer cells and immune cells, flow cytometry and cell sorting", "section_num": null }, { "section_content": "For IHC staining, the tumor tissues from patients or mice were fixed in formalin, dehydrated with graded alcohols, and then embedded in paraffin.Sectioned samples were deparaffined in xylene and rehydrated.Endogenous peroxidase was inactivated using 3% H 2 O 2 diluted in methanol, and antigen retrieval was performed with citric acid under high temperature and high pressure.The sections were then blocked with animal nonimmune serum (Maixin Biotech) and incubated with primary antibodies at 4 °C overnight.After washing, the sections were incubated with secondary antibodies at RT for 30 min and stained with DAB detection kit (MaxVision).Hematoxylin (ZSGB-BIO) was used for cell nucleus staining, and the visualization was achieved by microscope (OLYMPUS BX43).For IF staining, the sectioned samples were basically subjected to the same experimental operations as above.DAPI (Invitrogen) was used for cell nucleus staining, and the images were observed and captured by confocal microscope (Zeiss LSM710).The antibody information was shown in Supplementary Table 3. ", "section_name": "Immunohistochemistry (IHC) and Immunofluorescence (IF) staining", "section_num": null }, { "section_content": "To evaluate the myeloid differentiation potential of bone marrow progenitor cells, GMPs and GPs (1000 cells/well) sorted from BM of Balb/c mice bearing 4T1 pSIN-/CCL20-overexpressing cell orthotopic allograft tumors, or sorted from BM of Balb/c mice bearing parental 4T1 cell orthotopic allograft tumors with rmCCL20 (10 ng/ ml, R&D systems) in the culture using MethoCult GF M3434 (STEMCELL Technologies) in 96-well ultra-low attachment plates (Corning) for 10 days.Fresh methylcellulose-based medium was added every 2-3 days.The colonies were identified and counted by microscope (Olympus IX73). ", "section_name": "Colony-forming unit assay", "section_num": null }, { "section_content": "Murine CCR6-siRNA and non-silencing scrambled control (SCR) siRNA were purchased from GenePharma Biotech.The sequences corresponding to the indicated siRNA were as follows: CCR6-siRNA, 5'-GUGUAUGAGAAGGAAGAAUAAdTdT-3', 3'-UUAUUCUU CCUUCUCAUACACdTdT-5'; SCR-siRNA, 5'-UUCUCCGAACGUGU-CACGUdTdT-3', 3'-ACGUGACACGUUCGGAGAAdTdT-5'.Cells were cultured in serum-free medium and transfected with the a mixture of siRNA and Lipofectamine TM 3000 Transfection Reagent (Invitrogen) according to the manufacturer's instructions.incubation at 37 °C for 4 h, fresh culture medium supplemented with 10% FBS was added to the cells and the appropriate treatment was performed. Mammosphere formation assay Murine breast cancer cells (200 cells/well) were mix-cultured with PMN-MDSCs sorted from tumors in 96-well ultra-low attachment plates (Corning) for 10 days.The MammoCult Human Medium kit (StemCell Technologies) supplemented with 4 μg/ml heparin (StemCell Technologies), 1 μg/ml hydrocortisone (Sigma), and 1% pen-strep antibiotic (Beyotime Biotechnology) was required.Fresh complete mammocult medium was added every 3-4 days.After culture was completed, spheres were collected by centrifugation at 500 rpm for 5 min and digested with 0.25% trypsin at 37 °C for 5 min.Single cells were centrifuged and resuspended for subsequent experiments.Flow cytometry was used to distinguish breast cancer cells and PMN-MDSCs with specific cell surface markers.The images of mammospheres were observed by microscope (Olympus IX73). Co-culture or mix-culture of PMN-MDSCs and breast cancer cells PMN-MDSCs (1 × 10 6 cells/well) were sorted from tumors and mix-cultured or co-cultured with the corresponding breast cancer cells (2 × 10 5 cells/well) in 6-well plates for 3 days.Transwell insert with pore size of 0.4 µm (Corning) was applied for cell co-culture.PMN-MDSCs and breast cancer cells were placed in the lower and upper chambers of the insert respectively.Meanwhile, PMN-MDSCs from tumors were cocultured or mix-cultured with scramble or CXCR2-knockdown 4T1 cells in the same method. In vitro limiting dilution assay The murine breast cancer cell lines 4T1 or Py8119 were treated with rmCXCL2 (10 ng/ml) or co-cultured with pSIN PMN or CCL20 PMN from pSIN-/CCL20-overexpressing 4T1 or Py8119 cell allograft tumors for 3 days.Then, 4T1 cells were seeded at a density of 5, 10, 20, 50, 100 cells per well and Py8119 cells were seeded at a density of 25, 50, 100, 250, 500 cells per well in 96-well ultra-low attachment plates with complete mammocult medium.Fresh culture medium was added every 3-4 days, and the mammosphere formation was observed after 10 days.The BCSC frequency was calculated by the Extreme Limiting Dilution Analysis (ELDA, http://bioinf.wehi.edu.au/software/elda). Soft agar colony formation assay Breast cancer cells (8000 cells/well) were resuspended in culture medium containing 0.3% low melting agarose (Sangon Biotech) and overlaid with 0.6% low melting agarose in 6-well plates.After incubation for 4-6 weeks, colonies were stained with 0.005% crystal violet, and then the number of colonies was analyzed. ", "section_name": "RNA interference", "section_num": null }, { "section_content": "For RNA sequencing (RNA-Seq), breast cancer cells and PMN-MDSCs were obtained from tumors by flow cytometry, and 4T1 cells were treated with rmCCL20 (10 ng/ml, R&D systems) or PBS for 3 days in vitro.Total RNA of sorted cells and cell lines were extracted with TRIzol reagent (Takara Bio).RNA-Seq libraries were established using the NEB Next Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs) and checked by quality control with 2100 Bioanalyzer (Agilent).Sequencing was performed on HiSeq3000 platform (Illumina).The RNA-Seq data were subjected to unsupervised clustering and transformed into heat maps.Enrichment pathway analysis of genes was compiled from both GSEA and Metascape databases, and a p value < 0.05 was considered statistically significant. ", "section_name": "RNA sequencing", "section_num": null }, { "section_content": "All data were presented as the mean ± SEM and analyzed by GraphPad Prism 8.0.Unless otherwise indicated, comparisons between two data groups were performed with unpaired, twotailed Student's t test.Two-way ANOVA was used for multiple comparisons.P values were considered statistically significant as follows: p < 0.05, p < 0.01, p < 0.001 and ***p < 0.0001. ", "section_name": "Statistical analysis", "section_num": null } ]	[ { "section_content": "This work was supported by The National Key Research and Development Program of China (2020YFA0112300); National Natural Science Foundation of China (82230103, 81930075, 82203399, 82073267); \"Ten Thousand Plan\" -National High-Level Talents Special Support Plan (WR-YK5202101); Program for Outstanding Leading Talents in Shanghai; Program for Outstanding Medical Academic Leader in Shanghai (2019LJ04); Program of Shanghai Academic/Technology Research Leader (20XD1400700); The innovative research team of high-level local university in Shanghai. ", "section_name": "ACKNOWLEDGEMENTS", "section_num": null }, { "section_content": "RNA-seq data profiles from this study have been deposited in the NCBI under accession code PRJNA911328.All other data supporting the results can be found in this paper and its supplementary materials.All other relevant data can be obtained from the corresponding authors upon request. ", "section_name": "DATA AVAILABILITY", "section_num": null }, { "section_content": "Conceptualization: S.L.L., R.Z., L.Z., L.X.Z., M.X.D., Y.Z.; Methodology: R.Z., M.X.D., J.C.L.T., F.K.L., Q.D.D., Y.Z., J.H.X., X.Y.H., J.J.D., J.X., D.D.S.; Investigation: R.Z., M.X.D., F.K.L.; Visualization: R.Z., M.X.D., F.K.L., Z.X.C., W.M., H.N.D.; Funding acquisition: S.L.L.; Project administration: S.L.L.; Supervision: S.L.L., L.Z.; Writingoriginal draft: R.Z., M.X.D., S.L.L., L.Z.; Writingreview & editing: R.Z., M.X.D., S.L.L., L.Z.All authors have read and approved the article. The online version contains supplementary material available at https://doi.org/10.1038/s41392-023-01337-3. The authors declare no competing interests. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "Conceptualization: S.L.L., R.Z., L.Z., L.X.Z., M.X.D., Y.Z.; Methodology: R.Z., M.X.D., J.C.L.T., F.K.L., Q.D.D., Y.Z., J.H.X., X.Y.H., J.J.D., J.X., D.D.S.; Investigation: R.Z., M.X.D., F.K.L.; Visualization: R.Z., M.X.D., F.K.L., Z.X.C., W.M., H.N.D.; Funding acquisition: S.L.L.; Project administration: S.L.L.; Supervision: S.L.L., L.Z.; Writingoriginal draft: R.Z., M.X.D., S.L.L., L.Z.; Writingreview & editing: R.Z., M.X.D., S.L.L., L.Z.All authors have read and approved the article. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41392-023-01337-3. ", "section_name": "ADDITIONAL INFORMATION Supplementary information", "section_num": null }, { "section_content": "The authors declare no competing interests. ", "section_name": "Competing interests:", "section_num": null } ]
10.3389/fnmol.2018.00225	NFIX Circular RNA Promotes Glioma Progression by Regulating miR-34a-5p via Notch Signaling Pathway	Objective: The present study aimed to explore the association between NFIX circular RNA (circNFIX) and miR-34a-5p in glioma. Furthermore, this study investigated the influence that circNFIX has on glioma progression through the upregulation of NOTCH1 via the Notch signaling pathway by sponging miR-34a-5p. Methods: We applied five methods, CIRCexplorer2, circRNA-finder, CIRI, find-circ and MapSplice2, to screen for circRNAs with differential expression between three glioma tissue samples and three paired normal tissue samples. The GSEA software was used to confirm whether significantly different pathways were activated or inactivated in glioma tissues. The binding sites between circNFIX and miR-34a-5p were confirmed by TargetScan. QRT-PCR and western blot were used to measure the relative expression levels of circNFIX, miR-34a-5p and NOTCH and identify their correlation in glioma. RNA immunoprecipitation (RIP) validated the binding relationship between circNFIX and miR-34a-5p, while the targeted relationship between NOTCH1 and miR-34a-5p was verified by a dual luciferase reporter assay. Cell viability and mobility were examined by a CCK-8 assay and wound healing assay, and a flow cytometry assay was employed to analyze cell apoptosis. The nude mouse transplantation tumor experiment verified that si-circNFIX exerted a suppressive effect on glioma progression in vivo. Results: Twelve circRNAs were differentially expressed between the tissue types. Of those, circNFIX was the sole circRNA to be overexpressed in glioma among the five methods of finding circRNAs. In addition, the Notch signaling pathway was considerably upregulated in tumor tissues compared with the paired normal brain tissues. It was determined that circNFIX acted as a sponge of miR-34a-5p, a miRNA that targeted NOTCH1. Downregulation of circNFIX and upregulation of miR-34a-5p both inhibited cell propagation and migration. Furthermore, a miR-34a-5p inhibitor neutralized the suppressive effect of si-circNFIX on glioma cells. Si-circNFIX and miR-34a-5p mimics promoted cell apoptosis. Moreover, it was demonstrated in vivo that si-circNFIX could suppress glioma growth by regulating miR-34a-5p and NOTCH1. Conclusion: CircNFIX was markedly upregulated in glioma cells. CircNFIX could regulate NOTCH1 and the Notch signaling pathway to promote glioma progression by sponging miR-34a-5p via the Notch signaling pathway. This finding provided a deeper insight into the function of circNFIX in human glioma cancer progression.	[ { "section_content": "NFIX Circular RNA Promotes Glioma Progression by Regulating miR-34a-5p via Notch Signaling Pathway INTRODUCTION Glioma, a tumor in the central nervous system, has a poor survival rate and high mortality (Zhang Y. et al., 2017).The latest report suggested that the incidence of glioma has increased from 5.9/100,000 people to 6.61/100,000 people between 1973 and 2016 with the application of improved radiological diagnosis (Lu et al., 2017).Despite multiple treatment options, cancer remains one of the leading causes of death worldwide.It is therefore imperative that further research be completed to determine novel molecular targets for enhanced cancer therapy (Wang R. et al., 2017). Circular RNA (circRNA), a non-coding RNA with considerable regulatory potency, has received increased attention from RNA researchers in recent years (Li et al., 2015).Recently, it has been found that many circRNAs are derived from protein-coding exons and are widely expressed in cancer cells (Salzman et al., 2013).Most circRNAs originate from exons and are located in the cytoplasm (Venø et al., 2015).CircRNAs are formed by back-splicing covalently joined 3'-and 5'-ends (Zhang X. et al., 2017) and play a role in several cellular functions, including protein binding, RNA transport, and the regulation of translation (Zhang X. et al., 2017).Recently, it has been suggested that circRNAs play a critical role in late-stage gastric cancer (Fang et al., 2017).However, the influence of circRNAs in glioma needs further investigation. MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating various cellular functions by transcriptional silencing (Bezerra and Latronico, 2014).For instance, Zhou et al. (2017) proposed that miR-224 could target SMAD4 to promote colorectal cell propagation.It has been reported that miR-203 downregulates RGS17 to inhibit cell growth in non-small cell lung cancer (Chi et al., 2017).A growing body of research indicates that miRNAs can play key regulatory roles in glioma, uncovering novel biomarkers for glioma therapy.For example, a study by Xu L. et al. (2017) also indicated that miR-543 acted as a tumor suppressor that could inhibit glioma development in vitro and in vivo.MiR-34a is an essential member of the miR-34 family (Xu H. et al., 2017).Increasing evidence suggested that miR-34a is important in the research of glioma.Silber et al. (2012) verified that miR-34a significantly affected the growth of proneural glioma cells in vitro and in vivo.Li et al. (2014) found that upregulation of miR-34a inhibited glioma cell viability and promoted apoptosis.Gao et al. (2013)'s study also provided sufficient evidence for miR-34a as a potentially useful factor for predicting the prognosis of glioma.The present study investigates in greater depth the role of miR-34a-5p in glioma. Recently, a report demonstrated that a crosstalk exists between the Notch signaling pathways and miRNA in the development and progression of tumors (Wang et al., 2010).The Notch signaling pathway exerts an important function in the progression of various types of cancers by regulating cell propagation, apoptosis, and differentiation (Androutsellis-Theotokis et al., 2006).NOTCH1 was downregulated in oral cancer cells to help gamma-secretase inhibitors inhibit the spread of cancer cell proliferation (Yao et al., 2007).Thirty percent of all non-small cell lung carcinoma cases have increased activity of the Notch signaling pathway, 10% of which are induced by a mutation in the NOTCH1 gene (Westhoff et al., 2009).Many reports have shown that the overexpression of NOTCH1 could inhibit cell apoptosis in many cancers, which suggests its importance (Miele and Osborne, 1999;Jundt et al., 2002).Furthermore, previous reports have revealed that miR-34 could inhibit tumors by regulating the Notch signaling pathway (Subramaniam et al., 2012).MiR-34a could regulate liver regeneration and development in rats via the Notch signaling pathway (Wang X. P. et al., 2017), and miR-34a suppressed breast cancer stem cells by suppressing the Notch signaling pathway (Kang et al., 2015).However, the specific functions of Notch signaling pathways in glioma remain elusive. In this study, we focused mainly on the mechanism by which the circRNA circNFIX regulated the Notch signaling pathway and, therefore, glioma progression by regulating miR-34a-5p.RNA-Seq data were analyzed through CIRCexplorer2, circRNAfinder, CIRI, find-circ and MapSplice2 to screen differentially expressed circRNAs in glioma tissues.Gene Set Enrichment Analysis (GSEA) was performed with data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.By combining in vivo and in vitro experiments, we confirmed that the Notch signaling pathway was activated in glioma tissues.We further examined the expression levels of circNFIX and miR-34a-5p in glioma tissues and cells and verified their association with glioma progression. ", "section_name": "", "section_num": "" }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "Five kinds of blob selector were used in the experiment, including CIRCexplorer2, circRNA-finder, CIRI, find-circ and MapSplice2.RNA-Seq revealed the presence and quantity of RNA in a biological sample at a given moment in time by means of next-generation sequencing (NGS), which could avert many limitations of other transcriptomic approaches.The high-throughput sequencing data for three glioma and paired normal brain tissues from three males were downloaded from the Gene Expression Omnibus (GEO) database 1 , a publicly available database.The series accession number was GSE86202, and the platform was GPL16791.Data quality control (QC) was assessed with FastQC.GENCODEv.19Gene Transfer Format file was used as a transcript reference (GENCODE annotation).We used RNA-Seq to match sequencing data to the genome and then extracted the undirected comparison of fragments, followed by recombination and comparison with the genome. ", "section_name": "RNA-Seq Data", "section_num": null }, { "section_content": "The DEseq2 package was used to analyze the differentially expressed circRNAs and mRNAs with the threshold set as \|log 2 FD\| > 1 and adjusted P < 0.05 (FD: fold change).Through the Venn intersection analysis, 12 common circRNAs with differential expression including circNFIX were identified in glioma tumor tissues.The 10 most significantly upregulated mRNAs including NOTCH1 and the 10 most significantly downregulated mRNAs were screened out using the pheatmap package. ", "section_name": "Gene Expression Profiles", "section_num": null }, { "section_content": "GSEA was performed using data from the KEGG database.The expression data of total normalized mRNAs were uploaded to GSEA v3.0 software.Based on the analysis of GSEA, we also employed the R language ''GSEABase'' package to perform data processing.We used the ggplot2, DOSE, ggjoy, and clusterProfiler packages to construct the dotplot and joyplot. ", "section_name": "Gene Set Enrichment Analysis", "section_num": null }, { "section_content": "All cell lines used in the experiment including normal astrocytes HA1800 and human glioma cell lines SF-539, SHG-44 and U87 were obtained from BeNa Culture Collection (Beijing, China).The cell lines were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin and then incubated in a humidified atmosphere containing 5% CO 2 at 37 • C. ", "section_name": "Cell Culture", "section_num": null }, { "section_content": "Si-circNFIX, miR-34a-5p mimics and miR-34a inhibitor were synthesized by GenePharma Co, Ltd. (Shanghai, China).Si-NOTCH1 (#AM16708) was purchased from Invitrogen.The sequence of si-circNFIX is CACACTCCGGGATGAGTTCCA.Glioma cells were placed into a 6-well plate at a concentration of 1 × 10 6 cells in each well and cultured at 37 • C until 90% confluence was reached.Transfections were performed using the Lipofectamine 2000 kit (Invitrogen) according to the manufacturer's instructions, and the transfection efficiency of the cells was detected after 24 h of incubation. ", "section_name": "Cell Transfection", "section_num": null }, { "section_content": "A biotin-labeled circNFIX probe (5 -CACCCGTTCATCGAGG CACTGCTG-3 -biotin) was generated by Sangon Biotech Inc. 1 https://www.ncbi.nlm.nih.gov/geo/(Shanghai, China).We performed RNA immunoprecipitation (RIP) experiments using the Magna RIP TM RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer's instructions.U87 cells were first fixed for 10 min using 1% formaldehyde and then lysed and sonicated.Following centrifugation, 50 µl of supernatant was retained and incubated with circNFIX-specific probesstreptavidin Dynabeads (Invitrogen) and blended at room temperature for 24 h.Having washed the Dynabeads-probes-circRNA mixture and incubated it with 200 µl of lysis buffer, we utilized proteinase K to reverse the formaldehyde crosslinking.Lastly, TRlzol was added to the mixture for RNA extraction. ", "section_name": "RNA Immunoprecipitation (RIP)", "section_num": null }, { "section_content": "The fragment from NOTCH1 containing the putative binding sites for miR-34a-5p was amplified by PCR, cloned in the firefly luciferase expression vector pMIR-REPORT (Invitrogen) and named NOTCH1-WT.To mutate the putative binding sites for miR-34a-5p in NOTCH1, the sequence of the putative binding site was replaced as indicated and was named NOTCH1-MUT.Before being stably transfected with the pMIR-REPORT-NOTCH1-WT and pMIR-REPORT-NOTCH1-MUT reporter vectors, together with the Renilla luciferase-expressing vector pRL-TK (Promega, Madison, WI, USA) and the miR-34a-5p mimic or NC using LipofectamineTM 2000 (Thermo Fisher, USA), U87 cells were placed into a 24-well plate at a density of 5 × 10 5 cells in each well.The relative luciferase reporter activity was detected at 48 h post-transfection. ", "section_name": "Luciferase Reporter Assay", "section_num": null }, { "section_content": "We used TRIzol (Invitrogen, USA) to isolate the total RNA from cells according to the instructions.Real-time quantitative PCR (qRT-PCR) was used to measure the expression of miR-34a-5p, circNFIX, and NOTCH1.We used a TaqMan TM Advanced miRNA cDNA Synthesis Kit (#A28007, Applied Biosystems) to amplify the miRNA.CircRNA and mRNA were amplified with SuperScript TM VILO TM cDNA Synthesis Kit (#11754250, Invitrogen).Total RNA was detected by qRT-PCR using DyNAmo ColorFlash SYBR Green qPCR Kit (#F416XL, Invitrogen).Relative quantification of mRNA expression was normalized by the 2 -∆∆Ct method, and GAPDH was used for normalization.All reactions were carried out in triplicate by GeneAmp TM PCR System 9700 (Applied Biosystems).The primers are manifested in Supplementary Table S1. ", "section_name": "QRT-PCR", "section_num": null }, { "section_content": "Radioimmunoprecipitation assay (RIPA) buffer was utilized to prepare whole-cell lysates.Equal amounts of total protein (30 mg) from cell lysates were loaded on a 6% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis, after which they were transferred to a polyvinylidene difluoride membrane (Millipore).We used an enhanced chemiluminescence western blotting detection system (Bio-Rad) for detection.Primary antibodies used were those against NOTCH1 (#ab52627, 1:1000, Abcam, Hong Kong, China), Jagged1 (#ab7771, 1:500, Abcam), Hes1 (#ab71559, 1:500, Abcam), Hes5 (#ab194111, 1:2000, Abcam), and HEY2 (#ab86010, 1:1000, Abcam).Goat anti-rabbit IgG secondary antibodies (ab7090, 1:2000, Abcam) were employed at 37 • C for 1.5 h.The full original images have been uploaded as Supplementary Figure S1. ", "section_name": "Western Blot", "section_num": null }, { "section_content": "A Cell Counting Kit-8 assay (CCK-8, Dojin, Japan) was used to investigate U87 cell propagation.Cells were inoculated into 96-well plates with a 100-µl suspension in each well.Cells were cultured in DMEM medium in a 5% CO 2 incubator for 24 h.The OD values at different time points (24 h, 48 h, 72 h, 96 h and 120 h) were measured at 450 nm using a microplate reader (Varioskan Flash, Thermo Fisher) after adding the CCK-8 solution. ", "section_name": "Cell Propagation Assay", "section_num": null }, { "section_content": "Cell apoptosis was determined by means of a flow cytometer (FACS Calibur, BD, USA) based on the manufacturer's guidelines.After a 48-h transfection, we used flow cytometry to analyze the apoptosis rate between the control group, NC, si-circNFIX, mir-34a mimics, miR-34a-5p inhibitor and si-circNFIX + miR-34a-5p inhibitor cells, which were then washed and resuspended.We used FACS Diva (BD, USA) to analyze experimental data, and every experiment was done in triplicate. ", "section_name": "Apoptosis Analysis", "section_num": null }, { "section_content": "Cells were cultured in 6-well plates for 24 h to form a confluent monolayer.We used a sterile pipette tip (200 ml) to scratch the monolayer, washed the cells twice with PBS and incubated the cells in serum-free DMEM medium.Plates were photographed by a microscope (200×, Olympus) at 0 h and 48 h after scratching at an identical location, respectively, with the width of the scratch measured.The wound area was calculated as width 48 h /width 0 h × 100%.All experiments were performed in triplicate. ", "section_name": "Wound Healing Assay", "section_num": null }, { "section_content": "We used 24-well chambers with an 8-µm pore size (Corning, USA) to study cell migration.A total of 5 × 10 4 cells in 250 µl of DMEM containing 0.2% FBS were placed into the upper chamber, and 500 µl of DMEM (10% FBS) was supplemented into the lower chamber, followed by incubation overnight.Then, cells on the upper side of the well were removed.We fixed the wells in methanol for 20 min and used crystal violet to stain the cells.For quantification analysis, we captured photographs of five random fields.Three identical replicates were performed. ", "section_name": "Transwell Migration Analysis", "section_num": null }, { "section_content": "We purchased 12 male nude mice (4-week-old) from Shanghai SLAC Experimental Animal Center (Shanghai, China).All the experiments were known and approved by the Ethics Committee of the First Hospital of Jilin University.These male nude mice were assigned to two groups equally (Mock, si-circNFIX).U87 cells transfected with si-circNFIX (2 × 10 6 cells) were injected into the right limbs of the mice (six mice in each group).After 35 days, all the mice were sacrificed, and then tumors were resected and collected. ", "section_name": "Tumor Xenograft", "section_num": null }, { "section_content": "All data analyses were performed using GraphPad Prism 6.0.The above experiments were carried out at least three times.Continuous data were documented as the mean ± standard deviation.The difference between two groups was analyzed by Student's t-test.A P value < 0.05 was indicative of statistical significance. ", "section_name": "Statistical Analysis", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "Five kinds of Blob Selector were used to single out the circRNA that we were interested in, and we found there were 12 common circRNAs that were differentially expressed (Figure 1A).The results of the heat map showed that the 12 circRNAs presented significantly differential expression in tumor tissues, in which circNFIX was confirmed to be overexpressed by all five software packages (Figures 1B-F).The above results suggested that circNFIX was significantly upregulated in glioma tissues.Dotplot displayed 16 significantly different pathways in tumor tissues, of which the Notch signaling pathway was found to be activated in glioma tissues (Figure 2A).Furthermore, Joyplot also further confirmed that the Notch signaling pathway was upregulated in tumor tissues (P.adjust < 0.05, Figure 2B).The results of the heat map showed 20 differentially expressed genes (DEGs) including 10 upregulated genes and 10 downregulated genes, of which NOTCH1 was remarkably overexpressed in glioma tissues in comparison with adjacent normal tissues (Figure 2C).By intersecting the screened DEGs with genes in the Notch signaling pathway, we identified that NOTCH1 was notably upregulated (Figure 2D).Overall, the Notch signaling pathway was activated in glioma tissues compared with paired normal brain tissues. ", "section_name": "CircNFIX Expression and KEGG Pathway Analysis", "section_num": null }, { "section_content": "It is well known that circRNAs function mainly as miRNA sponges to regulate gene expression.We next found the potential miRNAs associated with circNFIX.The result in Figure 3A shows that several miRNAs containing binding sites with circNFIX existed; these miRNAs included miR-34a-5p, miR-526b, miR-646, miR-502-5p, miR-769-5p, miR-620, miR-874, miR-758-3p and miR-145-5p.Of the above miRNAs, miR-34a-5p was the closest to the 5'-UTR, drawing our interest.The binding sites between circNFIX and miR-34a-5p were validated by TargetScan (Figure 3B).Furthermore, in order to verify the relationship between circNFIX and miR-34a-5p in glioma cells, a RIP experiment was performed and confirmed that there was a specific enrichment of circNFIX and miR-34a-5p compared to the controls (Figure 3C).The qRT-PCR results showed that circNFIX was highly expressed in glioma cell lines SF539, SHG-44 and especially U87 cells compared to the normal astrocytes HA1800 (P < 0.01, Figure 4A).Meanwhile, the qRT-PCR results indicated that miR-34a-5p was significantly downregulated in glioma cell lines (P < 0.01, Figure 4B).Then, we selected the U87 cell line for the following experiments.After transfection with si-circNFIX, the expression of circNFIX was conspicuously decreased, while miR-34a-5p expression was considerably increased compared with the NC group (P < 0.01, Figures 4C,D).Additionally, TargetScan revealed that NOTCH1 3'-UTR WT had a binding site for hsa-miR-34a-5p, while NOTCH1 3'-UTR MUT could not bind to hsa-miR-34a-5p (Figure 4E).Moreover, the luciferase reporter assay also indicated that miR-34a-5p mimics remarkably repressed the relative luciferase activity of NOTCH1 WT but not NOTCH1 MUT in comparison with the miR-NC group (P < 0.01, Figures 4E-F).Taken together, these findings indicate that circNFIX was upregulated in glioma cells and the expression of miR-34a-5p was significantly increased by knockdown of circNFIX. ", "section_name": "Validation of Relationships Among circNFIX, miR-34a-5p and NOTCH1", "section_num": null }, { "section_content": "The qRT-PCR results suggested that NOTCH1 was overexpressed in glioma cells compared with the HA1800 cell line, especially in U87 cells (P < 0.01, Figure 5A).Si-circNFIX and miR-34a-5p mimics suppressed the expression of NOTCH1, while the miR-34a-5p inhibitor promoted the expression of NOTCH1.Si-circNFIX could reverse the facilitative effects of the miR-34a-5p inhibitor on NOTCH1 expression (P < 0.05, Figure 5B).Similarly, the western blot results suggested that miR-34a-5p mimics and si-circNFIX suppressed the expression of the NOTCH1 protein, while the miR-34a-5p inhibitor promoted the expression of the NOTCH1 protein (P < 0.05, Figure 5C).The downstream proteins Jagged1, Hes1 and HEY2 in the Notch signaling pathway were significantly downregulated in U87 cells after knocking down circNFIX, while the protein level of Hes5 was increased.It is probable that Notch-Hes signaling may have different impacts, depending on the glioma cell type or differentiation stage of the precursor cell (Wu et al., 2003; P < 0.01, Figure 5D).These results suggested that circNFIX could regulate NOTCH1 at both the mRNA and protein levels by acting as a sponge for miR-34a-5p. ", "section_name": "Si-circNFIX and miR-34a-5p Mimics Inhibited the Expression of NOTCH1 and the Downstream Protein Expression in the Notch Signaling Pathway", "section_num": null }, { "section_content": "The wound healing assay is often used to estimate the coordinated movement of a cell population (Rodriguez et al., 2005).The results of the wound healing suggested that si-circNFIX and miR-34a-5p mimics could inhibit cell migration and propagation, the miR-34a-5p inhibitor could promote wound healing, and the miR-34a-5p inhibitor could alleviate the suppressive effect of si-circNFIX on cell propagation (Supplementary Figure S2).The migration assay showed that si-circNFIX and miR-34a-5p mimics suppressed cell migration compared with the NC condition.The miR-34a-5p inhibitor could alleviate the suppressive impact of si-circNFIX (P < 0.05, Figures 6A,D).As the flow cytometry assay showed, si-circNFIX and miR-34a-5p mimics could promote cell apoptosis compared with NC.Similarly, miR-34a-5p could alleviate the suppressive impact of si-circNFIX (P < 0.05, Figures 6B,E).The CCK8 assay results suggested that the si-circNFIX and miR-34a-5p mimics suppressed cell proliferation and miR-34a-5p inhibitor promoted cell proliferation (P < 0.05, Figure 6C).All above results demonstrate that downregulation of circNFIX and overexpression of miR-34a-5p could suppress cell propagation and promote apoptosis. ", "section_name": "Si-circNFIX and miR-34a-5p Mimics Inhibited Cell Migration, Proliferation and Promoted Cell Apoptosis", "section_num": null }, { "section_content": "All above experiments demonstrated that si-circNFIX could regulate NOTCH1 to promote glioma progression by sponging miR-34a-5p in vitro.To confirm that knockdown of circNFIX could suppress the tumor growth of glioma in vivo, U87 cells transfected with si-circNFIX (2 × 10 6 cells) were injected into the right limbs of mice.Then, we found that the tumor volume and tumor weight containing si-circNFIX was significantly lower than those containing NC (Figure 7A).Then, the tumor tissues taken out from mice were used to detect the expression of circNFIX, miR-34a-5p and NOTCH1.The qRT-PCR results showed that the expression of circNFIX and NOTCH1 was significantly reduced in the si-circNFIX group compared with NC group, while miR-34a-5p was remarkably upregulated after treatment with si-circNFIX.These results indicated that si-circNFIX could repress the expression of circNFIX and NOTCH1 but promoted the expression of miR-34a-5p (P < 0.01, Figures 7B-D).Western blotting showed that circNFIX suppressed the expression of the NOTCH1 protein relative to that in the NC group (P < 0.01, Figure 7E).The results suggested that si-circNFIX exerted an inhibitory influence on glioma progression in vivo. ", "section_name": "Si-circNFIX Inhibited the Tumor Growth of Glioma in Vivo", "section_num": null }, { "section_content": "RNA-Seq, as a revolutionary tool for transcriptomics, has been applied in many experiments.For example, Mortazavi et al. (2008) predicted fusion transcripts based on the gene fingerprint profiles of the RNA-Seq paired-end reads (Li et al., 2017).Trapnell et al. (2009) discovered splice junctions with RNA-Seq. Until recently, circRNAs have largely been considered insignificant by the scientific community due to low expression levels (Chen et al., 2016).However, several research breakthroughs have provided profound evidence that they play major roles in biological functions.For example, evidence suggests that they play a key role in gene regulation, suggesting that they could make viable therapeutic targets in diseases such as cancer (Liu et al., 2016).Therefore, circRNAs have received significantly greater attention in recent years in the study of cancer.For example, Guarnerio et al. (2016) discovered that fusion-circRNAs could both allow cellular transformation and promote cell proliferation and had a tumorpromoting impact in cancer.Zhang H. et al. (2017) found that circRNA UBAP2 (circUBAP2) expression was increased in human osteosarcoma tissues.Li et al. (2018) identified that circ_0046701 was overexpressed in glioma.The study conducted by Yang et al. (2016) also clarified that knockdown of cZNF292 circular RNA could suppress tube formation by inhibiting glioma cell proliferation.Much is still unknown about the exact roles circRNAs may play in glioma development.Thus, we completed an RNA-Seq analysis comparing circRNA expression between glioma and matched adjacent normal tissue to better understand their role in glioma cells.Five methods, including CIRCexplorer2, circRNA-finder, CIRI, find-circ and MapSplice2, were used to screen out differentially expressed circRNAs.Via a Venn diagram, we found circNFIX was upregulated in all five methods.The focus of our project is to determine the role circNFIX plays in glioma progression in addition to uncovering the underlying mechanisms that drive it. As some of the noncoding RNAs, especially miRNAs, can be biomarkers for glioma diagnosis and treatment (Ahir et al., 2017), we also studied the function of a miRNA (miRNA-34a-5p) that played a role in glioma progression affected by circNFIX.Numerous studies have reported an important role of miR-34a in inhibiting various cancers, and hence, it is often deemed as a tumor suppressor gene (Chen et al., 2015).For instance, Guessous et al. (2010) confirmed that miR-34a was downregulated in glioma tumors.Gao et al. (2013) found that glioma tissues had lower miR-34a expression than normal brain tissues did.In our current study, the expression level of miR-34a-5p was also downregulated in glioma, which suppressed the expression of the genes downstream of the signaling pathway.A better understanding of the mechanism of circRNAs and miRNAs acting on glioma is meaningful for the discovery of effective therapies against glioma.Specifically, a large number of circRNAs have been verified to sponge miRNAs, thereby functioning as miRNA inhibitors.Bezzi et al. (2017) proposed that circRNA could function as a competing endogenous RNA, in which different RNAs could regulate each other by means of competing for miRNAs through miRNA recognition elements.As Han et al. (2017) reported that the circRNA circMTO1 acted as a sponge of microRNA-9 to repress liver cancer progression.Liang et al. (2017) confirmed the sponge effect of circ-ABCB10 on miR-1271 in breast cancer.Militello et al. (2017) also verified that in addition to lncRNAs, circRNAs could also function as miRNA sponges.These results were consistent with our findings.In our study, circNFIX could suppress glioma progression by sponging miR-34a-5p.However, recent studies had questioned the sponge role that circRNAs play on miRNAs and reported that circRNAs could be translated or take part in tumor progression through a variety of methods.For instance, Legnini et al. (2017) and Pamudurti et al. (2017) reported that circRNAs could function as a eukaryotic endogenous circular RNAs and encode proteins.This may be due to their property that limits the election of the target region for duplication and further restriction of the parameters commonly used for designing mRNA-targeting siRNAs.In this study, we investigated circNFIX expression and its potential role as a microRNA sponge in vitro and in vivo, further supporting the sponge role of circRNAs. Furthermore, we discovered that miR-34a-5p suppressed glioma progression in vitro by targeting the Notch signaling pathway, which was consistent with results that Jin et al. (2017) reported.In addition, Ji et al. (2008Ji et al. ( , 2009) ) found that miR-34 played an important role in cancer stem cell maintenance and survival.The above results all demonstrated that miR-34 could effectively regulate the Notch signaling pathway. However, this study has several limitations.First, our study was based on U87 cells.Though the U87 cell line is a representative glioma cell type, primary glioma cells derived from patients will be used in our future research.Second, additional mechanisms of circNFIX in regulating glioma progression require further study. Our research into the oncogenic role of circNFIX in glioma progression has uncovered possible mechanisms through which circRNAs influence cancer growth.However, due to the limited number of samples used in this study, we fully acknowledge that there may be additional circRNAs that play a key role that were not discovered in our data.However, our results suggest that the study of dysregulated circRNAs in cancer can prove fruitful to better understanding how to enhance cancer therapies. ", "section_name": "DISCUSSION", "section_num": null } ]	[ { "section_content": "We express our deep gratitude to the National Natural Science Foundation of China, the Foundation of Science and Technology Department of Jilin Province and the Project of Education Department of Jilin Province for their funding support. ", "section_name": "ACKNOWLEDGMENTS", "section_num": null }, { "section_content": "FUNDING This study was supported by National Natural Science Foundation of China (81201671); Foundation of Science and Technology Department of Jilin Province (20180101306JC, 20140101114JC, 20140204042YY, 2016C049-2); Project of Education Department of Jilin Province (2016236). ", "section_name": "", "section_num": "" }, { "section_content": "HX contributed to the study design.YZ and LQ contributed most to experiments practice and data collection.LD and HY helped to analyze data and HJ mainly wrote the manuscript.All authors contributed to the revision and checked the manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2018.00225/full#supplementary-materialFIGURE S1 \| Full original images of western blots.The expressions of NOTCH1, HEY2, Hes5, Hes1 and Jagged1 were accessed by western blot. The wound healing assay analyzed cell proliferation after the transfections.* P < 0.05 compared with the NC group, # P < 0.05 compared with the si-circNFIX group. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "HX contributed to the study design.YZ and LQ contributed most to experiments practice and data collection.LD and HY helped to analyze data and HJ mainly wrote the manuscript.All authors contributed to the revision and checked the manuscript. ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null }, { "section_content": "The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2018.00225/full#supplementary-materialFIGURE S1 \| Full original images of western blots.The expressions of NOTCH1, HEY2, Hes5, Hes1 and Jagged1 were accessed by western blot. ", "section_name": "SUPPLEMENTARY MATERIAL", "section_num": null }, { "section_content": "The wound healing assay analyzed cell proliferation after the transfections.* P < 0.05 compared with the NC group, # P < 0.05 compared with the si-circNFIX group. ", "section_name": "FIGURE S2 \|", "section_num": null }, { "section_content": "The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ", "section_name": "Conflict of Interest Statement:", "section_num": null } ]
10.1186/s12943-019-1084-1	Mettl14 inhibits bladder TIC self-renewal and bladder tumorigenesis through N6-methyladenosine of Notch1	<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p><jats:italic>N</jats:italic><jats:sup>6</jats:sup>-methyladenosine (m<jats:sup>6</jats:sup>A) emerges as one of the most important modification of RNA. Bladder cancer is a common cancer type in developed countries, and hundreds of thousands of bladder cancer patients die every year.</jats:p> </jats:sec><jats:sec> <jats:title>Materials and methods</jats:title> <jats:p>There are various cells in bladder tumor bulk, and a small population cells defined as tumor initiating cells (TIC) have self-renewal and differentiation capacities. Bladder TICs drive bladder tumorigenesis and metastasis, and their activities are fine regulated. However, the role of <jats:italic>N</jats:italic><jats:sup>6</jats:sup>-methyladenosine in bladder TIC self-renewal is unknown.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>Here, we found a decrease of <jats:italic>N</jats:italic><jats:sup>6</jats:sup>-methyladenosine in bladder tumors and bladder TICs. <jats:italic>N</jats:italic><jats:sup>6</jats:sup>-methyladenosine levels are related to clinical severity and outcome. Mettl14 is lowly expressed in bladder cancer and bladder TICs. Mettl14 knockout promotes the proliferation, self-renewal, metastasis and tumor initiating capacity of bladder TICs, and Mettl14 overexpression exerts an opposite role. Mettl14 and m<jats:sup>6</jats:sup>A modification participate in the RNA stability of Notch1 mRNA. Notch1 m<jats:sup>6</jats:sup>A modification inhibits its RNA stability. Notch1 plays an essential role in bladder tumorigenesis and bladder TIC self-renewal.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>This work reveals a novel role of Mettl14 and <jats:italic>N</jats:italic><jats:sup>6</jats:sup>-methyladenosine in bladder tumorigenesis and bladder TICs, adding new layers for bladder TIC regulation and <jats:italic>N</jats:italic><jats:sup>6</jats:sup>-methyladenosine function.</jats:p> </jats:sec>	[ { "section_content": "Bladder cancer is a serious cancer in the world, especially in advanced countries [1].There are many kinds of cells in bladder tumor, including bladder cancer stem cells (CSC), or tumor initiating cells (TIC) [2].Bladder TICs, a small population of cells in bladder tumors, have selfrenewal, differentiation and tumor-initiating capacities [2].Recently, increasing markers of bladder TICs were identified, and CD44 is one of the most widely-accepted markers [3].Compared with CD44 -cells, CD44 + cells show enhanced self-renewal and tumor-initiating capacities.Bladder TICs escape anoikis and initiate oncospheres in FBS-free medium, but bladder non-TICs can't survive [4].Accordingly, sphere formation assay emerges as one of the most important assays to detect bladder TIC selfrenewal.Besides sphere formation, transwell invasion assay can also be used for bladder TICs because of the critical role of bladder TICs in bladder metastasis and invasion [2,5].Like TICs in many other tumors, bladder TICs harbor enhanced tumor-initiating capacities, which can be examined by gradient tumor initiating assay [6][7][8].Highly expressing ABCG2 and other pump molecules, TICs are resistant to drug treatment [9].Despite of the critical role of bladder TICs in bladder tumor formation, metastasis, drug resistance and recurrence, the biological characteristics of bladder TICs are largely unknown. Like TICs in other tumors, bladder TICs are fine regulated, and the precise regulations of bladder TIC selfrenewal are still largely unknown [10,11].N 6 -methyladenosine (m 6 A) is the most abundant modification of mRNA in human and mice, and is very conserved among species [12].m 6 A modification is identified in plants, yeast, insects, virus and so on.Recently, m 6 A modification was also found on some non-coding RNAs, including tRNA, rRNA, lncRNA and snRNA [13].m 6 A modification is reversible, which is added by methyltransferase complex and removed by m 6 A demethylases.m 6 A methyltransferase complex is comprised of METTL3, METTL4, METTL14, WTAP, VIRMA and so on [14,15].On the contrary, FTO and AlkBH5 induce m 6 A demethylases [16,17].The influence of m 6 A in RNA stability is dependent on m 6 A readers, including YTHDF1, YTHDF2, YTHDF3 and YTHDC1 [18].m 6 A modification participates in many biological processes, including spermatogenesis and embryonic development [19][20][21], circadian period [22], DNA damage [23], hematopoietic stem cells [24] and tissue homeostasis [25].As for tumor biology, m 6 A modification exerts its role in tumorigenesis, proliferation and metastasis.Mettl14 is lowly expressed in hematopoietic stem cells and liver tumor cells.m 6 A methyltransferase Mettl14 attenuates the tumorigenesis of AML [26].Recently the inhibitory role of Mettl14 in liver tumorigenesis and metastasis was also revealed.Demethylase FTO drives tumorigenesis of acute myeloid leukemia [27].Another demethylase, ALKBH5 also exerts an oncogenic role in glioblastoma tumorigenesis [11].However, the roles of N 6 -methyladenosine and related enzymes in bladder tumorigenesis and bladder TIC self-renewal are largely unknown. Here, we found that m 6 A modification and Mettl14 were lowly expressed in bladder tumorigenesis and bladder TICs.m 6 A level and Mettl14 expression were negatively related to the bladder cancer severity and clinical outcome.m 6 A modification and Mettl14 inhibited bladder tumorigenesis and bladder TIC self-renewal through Notch1 signaling, adding a new layer of bladder TIC regulation and m 6 A function. ", "section_name": "Introduction", "section_num": null }, { "section_content": "", "section_name": "Materials and methods", "section_num": null }, { "section_content": "Anti-β-actin (cat.no.A1978) and DAPI (cat.no.28718-90-3) were obtained from Sigma-Aldrich.Anti-m 6 A antibody was purchased from Synaptic Systems.Anti-Mettl14 (ab220030) antibody and m6A quantification kit (ab185912) were from Abcam.Fluorescence-conjugated secondary antibodies were obtained from Molecular Probes Life Technologies. Primary bladder cancer samples were obtained from the Departments of Urology and Henan Institute of Urology, The First Affiliated Hospital of Zhengzhou University with informed consent.The details for bladder tumors used in this work were: # ", "section_name": "Reagents and samples", "section_num": null }, { "section_content": "Primary bladder cancer cells were obtained from bladder cancer patients.For primary cell isolation, a portion of excised tumor was incubated in Hanks balanced salt solution (HBSS; Gibco) and transported quickly to the laboratory on ice.Then the samples were cut into small fragments, and digested in HBSS containing 0.1% type I collagenase, 0.05% type IV collagenase, 0.03% pronase, and 0.01% deoxyribonuclease at 37 °C for 30 min.The sample was filtered through 70-μm-nylon filter and centrifuged for 4 min at 50 x g in 4 °C.Bladder cancer primary cells were in precipitation, and cell survival & purification were examined.For bladder TIC enrichment, bladder cancer primary cells were incubated with CD44 antibody for FACS, and TICs were used for sphere formation assay, m6A detection and other assays. ", "section_name": "Primary cell isolation and culture", "section_num": null }, { "section_content": "Sphere formation assay was detected to examine the self-renewal of bladder TICs.500 CD44 + bladder TICs were cultured in FBS-free DMEM/F12 medium (supplemented with 1 × B27 supplement, 1 × N2 supplement, 20 ng/ml bFGF and 20 ng/ml EGF) and seeded into Corning® Costar® Ultra-Low Attachment Multiple Well Plate (cat.no.3471, Corning).Two weeks later, sphere images were taken and sphere numbers were counted.For cell line detection, 1000 T24 cells were used for sphere formation. ", "section_name": "Sphere formation", "section_num": null }, { "section_content": "Mettl14 and Notch1 knockout cells were constructed through CRISPR/Cas9 approach.For knockout, sgRNAs were designed according to online tool (http://crispr.mit.edu/) and cloned into lentiCRISPRv2, which were transfected into 293 T cells for lentivirus package, and the lentivirus was concentrated with PEG-it Virus Precipitation Solution (System Biosciences).Bladder cancer primary cells were infected with lentivirus, and the transfected cells were collected by puromycin selection.Knockout efficiency was confirmed by Western blot, and then used for sphere formation assay, transwell invasion assay or other functional assays. ", "section_name": "CRISPR/Cas9 knockout", "section_num": null }, { "section_content": "For immunohistochemistry, 5-μm bladder cancer sections were treated with xylene (10 min × 2), 100% alcohol (5 min × 2), 95% alcohol (5 min), 75% alcohol (5 min), PBS (5 min), 3% H2O2 (20 min) and then boiled in Tris/EDTA buffer (PH 9.0) for antigen retrieval (20 min).Then the samples were incubated with anti-m 6 A (1:500 dilution in PBS) or anti-Mettl14 (1:500 dilution in PBS) antibodies for 2 h.After washing three times, HRP-conjugated secondary (1:500 dilution in PBS) and 3,3′-diaminobenzidine were used for visualization. ", "section_name": "Immunohistochemistry", "section_num": null }, { "section_content": "For m 6 A dot blot, RNA were extracted from bladder tumor, TICs and spheres using standard Trizol method, and then spotted onto nylon membrane.The samples were crosslink with UV treatment, and followed by m 6 A antibody incubation (1:2000 dilution in PBS, supplemented with 5% milk) and subsequent HRP-conjugated secondary antibody (1:5000 dilution in PBS, supplemented with 5% milk), finally the samples were detected with 3,3′diaminobenzidine.For loading control, 0.02% methylene blue was used to stain the same RNA samples. ", "section_name": "Dot blot", "section_num": null }, { "section_content": "For FACS sorting or detection, samples were incubated with Phycoerythrin (PE)-conjugated CD133 (1:300 dilution in FACS buffer) or control antibodies (1:300 dilution in FACS buffer) for 30 min on ice, and then subjected to FACS.For FACS sorting, CD133 + bladder TICs and CD133 -non-TICs were enriched.For detection, FlowJo software (FlowJo v10) was used for data analyses. ", "section_name": "FACS", "section_num": null }, { "section_content": "For bladder TIC ratio analysis, 10, 1 × 10 2 , 1 × 10 3 , 1 × 10 4 and 1 × 10 5 cells were injected into BALB/c nude mice for three months' tumor formation.The ratios of bladder TICs were calculated by ELDA (extreme limiting dilution analysis) with online software (http://bioinf.wehi.edu.au/software/elda/).For most experiments, two tailed unpaired Student's t-test was used for statistical analysis. ", "section_name": "Statistical methods", "section_num": null }, { "section_content": "", "section_name": "Results", "section_num": null }, { "section_content": "As the most widely distributed RNA modification in mammalian cells, m 6 A modification exerts critical roles in many biological processes.However, its role in bladder tumorigenesis and bladder TICs is unknown.In this work, we focused on the role of m 6 A modification in bladder tumorigenesis and bladder TICs, and we detected the content of m 6 A modification in bladder tumor first.m 6 A modification was detected in non-tumor, early and advanced bladder tumors, and decreased m 6 A content was observed along with bladder tumorigenesis.Moreover, m 6 A modification was related to clinical severity (Fig. 1a).The reduction of m 6 A modification in bladder cancer was also validated by RNA dot blot (Fig. 1b), immunohistochemistry (Fig. 1c) and bladder cancer tissue array (Fig. 1d,e).As expected, lower content of m 6 A modification was also detected in advanced tumors by bladder cancer tissue array and immunohistochemistry (Fig. 1e).Moreover, m 6 A modification was also related to the clinical outcome of bladder tumor patients (Fig. 1f).Taken together, m 6 A modification content was lower in bladder tumors and related to clinical severity. ", "section_name": "Decreased content of m 6 A modification in bladder cancer", "section_num": null }, { "section_content": "To further examine m 6 A modification in bladder TICs, we enriched CD44 + bladder TICs and CD44 -non-TICs, and detected m 6 A modification.Compared with non-TICs, lower m 6 A modification levels were found in bladder TICs (Fig. 2a), which was confirmed by RNA dot blot and immunofluorescence (Fig. 2b,c). TICs can survive in sphere formation medium and sphere formation assay emerges as a standard method to enrich bladder TICs.Accordingly, sphere formation assay was performed and spheres were collected to detect m 6 A modification.Compared with non-spheres, lower m 6 A modification levels were found in oncospheres (Fig. 2d).RNA dot blot and immunofluorescence also confirmed the decreased content of m 6 A modification in bladder cancer spheres (Fig. 2e,f).Meanwhile, lower m 6 A modification was also observed in T24 spheres (Fig. 2g).Taken together, m 6 A modification was decreased in bladder TICs. ", "section_name": "Decreased m 6 A modification in bladder TICs", "section_num": null }, { "section_content": "Considering the importance of methyltransferases, demethylases and m 6 A readers in m 6 A modification, we then detected the expression profiles of m 6 A-related genes.Among these genes, Mettl14 was lowly expressed in bladder cancer, especially in advanced bladder cancer samples (Fig. 3a).What's more, lower expression of Mettl14 was detected in bladder TICs and spheres (Fig. 3b).To further examine Mettl14 expression pattern, bladder cancer tissue array was performed and confirmed the lowly expression of Mettl14 in bladder cancer (Fig. 3c).Interestingly, Mettl14 was also lowly expressed in advanced bladder tumors and related to the prognosis of bladder tumor patients (Fig. 3d,e). To further confirm the role of Mettl14 in m 6 A modification, we generated Mettl14 knockout cells through CRISPR/Cas9 approach (Fig. 3f).Mettl14 knockout led to decreased content of m 6 A modification, indicating the critical role of Mettl14 in m 6 A modification (Fig. 3g,h).Moreover, a positive correlation of Mettl14 expression and m 6 A content was observed in bladder tumors (Fig. 3i).What's more, in vitro RNA N 6 -adenosine methylation assay also confirmed the activity of Mettl14 in RNA m 6 A modification (Fig. 3j).Altogether, Mettl14 was lowly expressed in bladder cancer and accounted for the decreased content of m 6 A modification. ", "section_name": "Mettl14 was lowly expressed in bladder TICs and accounted for m 6 A suppression", "section_num": null }, { "section_content": "To further explore the role of Mettl14 in bladder tumorigenesis and bladder TICs, we utilized Mettl14 knockout cells to perform sphere formation assay. Mettl14 knockout cells showed increased capacity of sphere formation, indicating the inhibitory role of Mettl14 in bladder TIC self-renewal (Fig. 4a).Moreover, Mettl14 knockout cells contained increased bladder TICs, which confirmed the inhibitory role of Mettl14 in bladder TIC maintenance (Fig. 4b).Cell proliferation was detected by Ki67 staining, and Mettl14 knockout cells showed enhanced proliferation capacity (Fig. 4c).Bladder TICs account for bladder tumor invasion and metastasis, and thus tumor invasion was also detected.Mettl14 knockout cells have enhanced invasion capacity, revealing the inhibition of Mettl14 in bladder tumor invasion (Fig. 4d,e).We then detected tumor propagation of Mettl14 knockout cells through in vivo tumor propagation assay.Increased tumor propagation was found in Mettl14 knockout cells (Fig. 4f). To further explore the role of Mettl14 in tumor initiation, Mettl14 knockout cells were used for tumor initiation assay in vivo.Gradient Mettl14 knockout cells were injected into BALB/c nude mice for tumor initiation.Mettl14 deleted cells showed enhanced tumor formation ability (Fig. 4g).Consist with tumor initiation, higher ratios of bladder TICs were also detected in Mettl14 knockout cells (Fig. 4h).Meanwhile, Mettl14 deleted T24 cells showed enhanced oncosphere formation capacity (Fig. 4i), enhanced invasion capacity (Fig. 4j) and tumor propagation ability (Fig. 4k).Altogether, Mettl14 knockout promoted the self-renewal of bladder TICs. ", "section_name": "Mettl14 knockout drove bladder TIC self-renewal", "section_num": null }, { "section_content": "We then overexpressed Mettl14 in bladder TICs and examined the self-renewal.Mettl14 overexpressing cells were generated through lentivirus and confirmed by Western blot (Fig. 5a).Mettl14 overexpression led to a decrease in sphere formation (Fig. 5b), cell proliferation (Fig. 5c) and TIC ratios (Fig. 5d).Meanwhile, Mettl14 overexpressing cells showed impaired role in tumor invasion (Fig. 5e,f) and propagation (Fig. 5g). Mettl14 overexpressing cells were also used for tumorinitiating assay, and impaired tumor initiation was observed (Fig. 5h).Decreased ratios of bladder TICs were also calculated through extreme limiting dilution analysis (Fig. 5i).Meanwhile, the inhibitory role of Mettl4 in bladder cancer sphere formation, invasion and tumor propagation was also confirmed in T24 cells (Fig. 5j-l).Altogether, Mettl14 overexpression blocked the selfrenewal of bladder TICs. ", "section_name": "Mettl14 overexpression inhibited TIC self-renewal", "section_num": null }, { "section_content": "Finally we explored the functional target genes in bladder TICs.We focused on the target genes of three major pathways, including Wnt/β-catenin, Notch and Hedgehog pathways, and found Notch1 was highly expressed in Mettl14 knockout cells, and lowly expressed in Mettl14 overexpressing cells (Fig. 6a).The inhibitory role of Mettl14 in Notch1 expression was confirmed by Western blot (Fig. 6b,c).Considering the role of m 6 A modification in mRNA stability [18,19], we examined the stability of Notch1 mRNA in Mettl14 knockout cells.Interestingly, enhanced stability of Notch1 was observed upon Mettl14 knockout (Fig. 6d).Moreover, a negative correlation of Mettl14 expression and Notch1 expression was observed in bladder tumors (Fig. 6e). Although the role of Notch signaling in TICs is wellknown, its role in bladder TICs remains elusive.To explore the role of Notch1 in bladder TICs and Mettl14 function, we generated Notch1 knockout cells through CRISPR/Cas9 approach (Fig. 6f).Compared with control cells, Notch1 knockout cells showed impaired sphere formation and invasion capacities, revealing the essential role of Notch1 in bladder TICs (Fig. 6g-j).More importantly, in Notch1 knockout cell, Mettl14 knockdown showed impaired role for sphere formation and metastasis, indicating the critical role of Notch1 in Mettl14 function (Fig. 6g-j).The essential role of Notch1 in Mettl14 function was confirmed in T24 bladder cancer cell line (Fig. 6k).Altogether, Mettl14 targeted Notch1 that was essential for bladder TICs. ", "section_name": "Mettl14 targeted Notch1 mRNA stability in bladder TICs", "section_num": null }, { "section_content": "As the most abundant modification in human mRNA, m 6 A modification participates in many physiological and pathological processes [28].However, its role in bladder tumorigenesis and bladder TICs is unknown.In this work, we discovered the low content of m 6 A modification in bladder tumorigenesis and bladder TICs (shown in Figs. 1 and2), and identified the role of m 6 A modification and Mettl14 through various functional assays, including sphere formation, transwell invasion assay, gradient tumor initiation assay, tumor propagation assay and Ki67 staining (shown in Figs. 4 and5).Our work defined Mettl14 as a tumor suppressor gene in bladder and a negative modulator in bladder TICs (shown in Figs. 3, 4 and5). The self-renewal of bladder TIC is precisely regulated, and the regulation mechanism is largely unknown.Here, we revealed a novel regulatory axis of bladder TICs.Mettl14 knockout drove the self-renewal of bladder TICs (shown in Fig. 4), and Mettl14 overexpression inhibited bladder TIC self-renewal (shown in Fig. 5).Mettl14 largely attenuated Notch1 expression, and participated in bladder TICs through Notch1 (shown in Fig. 6).Mettl14-m 6 A-Notch1 pathway plays a critical role in bladder tumorigenesis and bladder TICs. (See figure on previous page.)Fig. 2 m 6 A modification was reduced in bladder TICs. a Bladder TICs and non-TICs were sorted by FACS with CD44 antibody, and mRNA was extracted for m 6 A detection.Six bladder tumors were used for TIC enrichment and subsequent m 6 A detection.b m 6 A RNA dot blot in bladder TICs and non-TICs.RNA extracted from bladder TICs and non-TICs was examined for m 6 A modification.Six samples were examined and got similar results.c FACS enriched TICs and non-TICs were stained with m 6 A and CD44 antibodies, and visualized by confocal microscopy.d Sphere formation was performed, followed by m 6 A detection using spheres and non-spheres.e Spheres and non-spheres were generated from bladder primary cells and m 6 A modification was detected.f, g Oncospheres and non-spheres were enriched from bladder cancer sample (f) or T24 cell line (g), followed by immunofluorescence detection of m 6 A modification.DIC, differential interference contrast.Six samples were examined and similar results were obtained, and sample #1 results were shown.P < 0.05, P < 0.01, P < 0.001, by two-tailed T test.At least three independent experiments were performed and got similar results Wnt/β-catenin, Notch and Hedgehog pathways were the most important signaling pathways in TICs of various tumors [29][30][31][32].We detected the related genes of Wnt/βcatenin, Notch and Hedgehog signaling, and identified Notch1 was a functional target gene of m 6 A modification and Mettl14 (Fig. 6a).Then we generated Notch1 knockout cell and re-evaluated the role of Mettl14.Impaired Mettl14 role was found upon Notch1 knockout, indicating Mettl14 mainly target Notch1 to inhibit bladder TIC (Fig. 6).As a common enzyme, there must be many target genes of Mettl14, but here we defined Notch1 was the functional target gene through loss of function assay.We think some other target genes may also involve in other biological processes, but in bladder tumorigenesis and bladder TIC self-renewal, Mettl14 exerted its role mainly through Notch1-dependent manner. TICs were regulated by various signaling pathways, including Wnt/β-catenin, Notch and Hedgehog pathways.Increasing works revealed the critical role of Wnt/β-catenin and Hedgehog pathway in bladder tumorigenesis and bladder TIC self-renewal.However, the role of Notch signaling in bladder tumorigenesis and TICs remains elusive.Here we generated Notch1 knockout cells and revealed an impaired activity of bladder TICs (shown in Fig. 6).Our work defined the oncogenic role of Notch1 in bladder cancer. The mRNA modification is a complicated process, with various modifications for various RNA molecules.Besides m 6 A, N 1 -methyladenosine, m 5 C and pseudouridine also emerge as critical modulators in various biological processes [33,34].m 6 A modification has been identified on mRNA and some non-coding RNA, including microRNA, lncRNA and snoRNA [35].Increasing evidences reveal that lncRNA emerges as critical modulators in the self-renewal of many kinds of TICs [36][37][38].The role of m 6 A and other modifications in non-coding RNAs also need to be further investigated. ", "section_name": "Discussion", "section_num": null }, { "section_content": "Bladder TICs drive bladder tumorigenesis and metastasis, and their regulation remains largely unknown.Here we revealed the role of N 6 -methyladenosine in bladder TIC self-renewal, adding new layers of bladder TIC regulation and N 6 -methyladenosine function.As a novel modulator of TICs, Mettl14-N 6 -methyladenosine-Notch1 pathway may be a potential target for bladder TIC elimination. (See figure on previous page.)Fig. 6 Mettl14 and m 6 A modification inhibited the stability of Notch1.a The related genes of Wnt/β-catenin, Notch and Hedeghog signaling were analyzed in Mettl14 knockout cells and Mettl14 overexpressing cells, and the expression was shown as heatmap.b, c Notch1 expression levels in Mettl14 knockout cells (b) and Mettl14 overexpressing cells (c) were examined by Western blot.Gapdh served as a loading control.d Mettl14 knockout cells were treated with 2 μg/mL actinomycin D, and then Notch1 mRNA levels at the indicated time points were examined by Northern blot.Actb was a loading control.e Correlation of Notch1 and Mettl14 expression.The expression levels of Notch1 and Mettl14 were used for analysis.Pearson correlation coefficient (R) and P-value were calculated.f Notch1 knockout cells were generated through CRISPR/Cas9 approach and examined by Western blot.g, h) Sphere formation of Notch1 knockout cells.For G, typical images were shown in upper panels and calculated numbers were shown in lower panels.For H, spheres were detected for m6A levels, confirming the decreased m6A levels upon Mettl14 knockdown.i, j Sphere formation of Notch1 knockout cells.For I, typical images were shown in left panels and calculated numbers were shown in right panels.For J, invasive cells were detected for m6A levels, confirming the decreased m6A levels upon Mettl14 knockdown.k Mettl14 was silenced in Notch1 knockout T24 cells, followed by sphere formation assay.*P < 0.001; ns, not significant, by two-tailed T test.At least three independent experiments were performed and got similar results ", "section_name": "Conclusion", "section_num": null } ]	[ { "section_content": "Not Applicable. This work reveals a novel role of Mettl14 and N6-methyladenosine in bladder tumorigenesis and bladder TICs, adding new layers for bladder TIC regulation and N6-methyladenosine function. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "Not Applicable. ", "section_name": "Acknowledgments", "section_num": null }, { "section_content": "This work reveals a novel role of Mettl14 and N6-methyladenosine in bladder tumorigenesis and bladder TICs, adding new layers for bladder TIC regulation and N6-methyladenosine function. ", "section_name": "Precis for use in the table of contents", "section_num": null }, { "section_content": "This work was supported by the National Natural Science Foundation of China (NO.81100464, 81200883 and 81570685); the National Natural Science Foundation of Henan (NO.2018061);theMedical Key Technologies R & D Program of Henan (201702031, 201702015); the Key Scientific Research Foundation of the Higher Education of Henan Province (No. 20A320032, 20A320044). ", "section_name": "Funding", "section_num": null }, { "section_content": "All data and materials can be provided upon request. ", "section_name": "Availability of data and materials", "section_num": null }, { "section_content": "Abbreviations CSC: Cancer stem cells; m6A: N6-methyladenosine; TIC: Tumor initiating cells Authors' contributions CG, performed experiments, analyzed data and wrote the paper; ZW, NZ, GL, YK, YL, and YW.performed some experiments; JY. and FT.initiated the study, designed experiments and wrote the paper.All authors read and approved the final manuscript. Primary bladder cancer samples were from the the First Affiliated Hospital of Zhengzhou University with informed consent. The authors agree for publication. The authors declare that they have no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Ethics approval and consent to participate", "section_num": null }, { "section_content": "Abbreviations CSC: Cancer stem cells; m6A: N6-methyladenosine; TIC: Tumor initiating cells Authors' contributions CG, performed experiments, analyzed data and wrote the paper; ZW, NZ, GL, YK, YL, and YW.performed some experiments; JY. and FT.initiated the study, designed experiments and wrote the paper.All authors read and approved the final manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "Primary bladder cancer samples were from the the First Affiliated Hospital of Zhengzhou University with informed consent. ", "section_name": "Ethics approval and consent to participate", "section_num": null }, { "section_content": "The authors agree for publication. ", "section_name": "Consent for publication", "section_num": null }, { "section_content": "The authors declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ", "section_name": "Publisher's Note", "section_num": null } ]
10.3747/co.25.3756	Improving Pneumococcal Vaccine Uptake in Veterans with Chronic Lymphocytic Leukemia through a Virtual Clinic	<jats:p>Through a “virtual clinic,” we used the electronic medical record to identify and intervene upon patients with chronic lymphocytic leukemia (CLL) who were not current for pneumococcal vaccines. Within 180 days, 100/160 patients (62%) received the recommended pneumococcal vaccine. A virtual clinic may improve vaccination rates among high-risk patient populations.</jats:p>	[ { "section_content": "Although pneumococcal immunizations are an important component of preventive health for adults, national immunization rates remain around 20%, well below the goal of 60% set by the United States Department of Health and Human Services 1,2 .For chronically ill adults who receive health care via specialty clinics, additional barriers may exist.First, physicians are both overly broad in applying contraindications and overestimate the proportion of patients in their panels who are current for adult vaccinations 3 .Second, specialty care clinics focus on specific medical problems and may overlook some aspects of preventive health, such as adult vaccinations.This may be due to competing priorities or because specialists believe that patients' primary care physicians will address vaccines and other preventive health measures 4 .Third, health system barriers including incomplete or inaccessible documentation of previous vaccines may also contribute to missed opportunities for vaccination 4 . The Advisory Committee on Immunization Practices (acip) recommends that adults with immunocompromising conditions, including chronic lymphocytic leukemia (cll), should receive one dose of the 13-valent pneumococcal conjugate vaccine (pcv13) and two doses, five years apart, of the 23-valent pneumococcal polysaccharide vaccine (ppsv23) 5 .Previously, we used a \"virtual clinic\" to improve rates of pneumococcal vaccine coverage among people with functional or anatomic asplenia 6 .Here, we describe using the virtual clinic to improve pneumococcal vaccinations among cll patients at a single Veteran Affairs (va) medical centre. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "Using structured language query (sql) to access our local Veterans Health Administration (vha) database, we employed International Classification of Diseases (icd) codes to identify a cohort of patients at our va medical centre with cll (icd9 codes 204.1 to 204.99 and icd10 codes C91.1, C91.90 and C91.Z).Inclusion criteria were a diagnosis of cll at the time of the study intervention and active in our regional medical system, defined as at least one in-person visit to a va clinic or hospital in our catchment area in the previous year.The exclusion criteria were a transfer of care to a va medical centre outside of our catchment area, a residential postal address outside of Ohio, actively receiving chemotherapy or hospice care, or a lack of clinic visits or medication refills in the prior year.Using both the vha database and the electronic medical record (emr), we further refined the cohort, verifying each patient's pcv13 and ppsv23 immunization status, including the number and time intervals for ppsv23. In August 2015, patients meeting our inclusion and exclusion criteria that were not current for either pcv13 and/or ppsv23 were enrolled into a \"virtual clinic\" as e96 Current Oncology, Vol. 25, No. 1, February 2018 © 2018 Multimed Inc. previously described 6 .The virtual clinic intervention had three components.First, virtual clinic providers reviewed the emr and placed an order for the appropriate pneumococcal vaccine that remained active for 90 days.Second, they wrote a note in the emr explaining the rationale for the vaccine and added as cosigners both the nurse and provider from the primary care patient-aligned care team (pact) working collaboratively to address the patients' needs.For those patients receiving care for their cll from the va, the nurse and providers from the oncology team were also included as cosigners.Finally, the virtual clinic providers mailed a letter to patients recommending they receive a pneumococcal vaccine, explaining that they could receive the vaccine at any va facility, and that the appropriate vaccine was ordered.The letters explained risks of pneumococcal infection, the benefits of vaccination, and included the appropriate vaccine information statements for pcv13 or ppsv23 7,8 . The primary outcome measured after the above intervention was the administration of the recommended vaccine within 180 days following the placement of the pneumococcal vaccine order.Additional outcomes, assessed via chart review, included the type of clinic in which patients received the vaccine or reasons why patients were not vaccinated. In May 2015, three months before this virtual clinic began, the va updated a national clinical reminder about pneumococcal vaccines to include recommendations for pcv13.To assess the effect of the virtual clinic on vaccination rates beyond that of the clinical reminder, we considered pcv13 vaccination rates before the clinical reminder (February -April 2015), after the introduction of the clinical reminder (May -July 2016), and after implementation of the virtual clinic (August -October 2016).For each of these periods, we considered the \"at-risk\" population to be active cll patients not yet vaccinated with pcv13.We used a log-rank test to assess differences in the pcv13 vaccination rates.All analyses were performed using R (R Version 3.1.1;Vienna, Austria) 9 .The Cleveland va Medical Center's Institutional Review Board (IRB) approved the research protocol. ", "section_name": "METHODS", "section_num": null }, { "section_content": "The initial sql query identified 336 patients with cll, 249 (74%) of whom were not current with their pneumococcal vaccines.After applying the exclusion criteria, 165 patients were enrolled in the virtual clinic.The majority were male (163; 99%) and self-identified as white (146; 88%); their mean age was 75 years (range 44 -98).Most of the patients needed only pcv13 (154; 93%).For the minority that needed both pcv13 and ppsv23 (11, 7%), virtual clinic providers recommended pcv13 first, in accordance with acip recommendations 10 . Figure 1 presents an overview of the outcomes.During the observation period, primary care providers determined that three patients had received pcv13 from a non-va provider prior to enrolment in the virtual clinic and added this information to those patients' charts.Additionally, two patients died, leaving 160 patients in the cohort at the end of the observation period.A total of 100 patients (62%) in the virtual clinic received the recommended pneumococcal vaccine within 180 days of notification.Of these patients, 90 (90%) received their vaccine through their va primary care pact, while the remaining 10 patients received them from va specialty clinics, non-va providers, or during hospitalization or home visits.Of the 60 patients who did not receive the recommended pneumococcal vaccine, the most common reasons were infrequent visits to healthcare providers (24, 40%) and patients declining vaccination (22, 37%). Figure 2 shows rates of pcv13 vaccination for previously unvaccinated cll patients at three consecutive threemonth intervals.Using a log-rank test, we compared the time-to-event curves for the interval immediately following the introduction of the clinical reminder and following the virtual clinic intervention and found them to be significantly different (p < 0.01).These outcomes indicate that the virtual clinic increased pcv13 vaccination rates beyond that achieved by the clinical reminder alone. ", "section_name": "RESULTS", "section_num": null }, { "section_content": "Our outcomes suggest that the virtual clinic strategy improves pneumococcal vaccination rates in a population that receives care through both primary and specialty care clinics.Furthermore, the increase in the vaccination rate following implementation of the virtual clinic indicates that this intervention augmented the clinical reminder in the emr. Previous work by Lau et al. indicates that patient outreach, education, and clinical reminders are effective strategies to improve pneumococcal vaccination 11 .Our virtual clinic coupled outreach with education by sending letters notifying patients of their need for and the importance of pneumococcal vaccination.The virtual clinic also brought the need for vaccination to the attention of the patients' FIGURE 1 Overview and outcomes of a virtual clinic for CLL patients at a VA medical center.The primary outcome was vaccination with the recommended pneumococcal vaccine in the 180-day observation period following the virtual clinic intervention.CLL = chronic lymphocytic leukemia; PCV = pneumococcal conjugate vaccine; VA = veteran affairs. primary care and oncology teams.Most patients in our cohort who received a pneumococcal vaccine did so via their primary care pact, which may reflect confidence in their providers, ease of access, and geographic proximity to their homes.Previous results indicated that notifying the pact nurses as well as the primary care provider improved vaccination rates 6 .In this intervention, some patients remained unvaccinated because they did not have medical visits during the observation period or declined the vaccine.Specific outreach from the primary care and/ or oncology teams to schedule a visit and provide education may have further increased vaccination rates. Clinical reminders incorporated within the emr may increase the likelihood of pneumococcal vaccination among patients at vha medical centers 12 .Our regional emr had a pneumococcal clinical reminder for ppsv23 in place for several years, which helps to explain the relatively high compliance with this vaccine among our cohort.Updated to include pcv13 three months before the virtual clinic intervention, the pneumococcal clinical reminder prompts providers to discuss pneumococcal vaccines with patients, permits documentation of vaccine refusal or acceptance, and guides appropriate vaccine selection.The clinical reminder, however, still requires providers to prioritize vaccinations in the context of other clinical care, discuss the vaccine with the patient, and to order the vaccine.An additional burden is \"alert fatigue\" as a result of the proliferation of clinical decision support notifications targeting various medical conditions and health measures 13 .The virtual clinic overcame one of these barriers by ordering the appropriate vaccine.Furthermore, notifying patients via letters mailed to their home may have made it easier for patients and providers to prioritize and discuss vaccination, in part by shifting some of this responsibility to the patient. Our study has limitations.First, the intervention focused on a small group of patients within a single medical centre.Second, we did not systematically assess the reasons why patients did not receive the recommended vaccine, nor did we probe into reasons why patients refused vaccination.Third, the clinical reminder that preceded the virtual clinic intervention influenced pneumococcal vaccination rates, confounding the impact of the virtual clinic.Changes in the pcv13 vaccination rates following the virtual clinic intervention, however, suggest that it enhanced pcv13 vaccination rates beyond that of the clinical reminder alone. The vha's emr and patient-centered infrastructure were both highly conducive to this virtual clinic intervention, which can be implemented in vas nationwide.Several components may also be generalized to other healthcare settings that also have a robust emr accessible by primary and specialty care services.Specifically, these include using data queries to identify high-risk patients who need vaccinations, alerting providers about those patients, and sending letters to patients encouraging them to make an appointment and discuss vaccines with their healthcare team.Approximately three months before the virtual clinic intervention, the vha deployed a system-wide pneumococcal vaccine clinical reminder.To assess that influence compared with the virtual clinic, we compared the rates of PCV13 vaccination over 90-day intervals before the clinical reminder (January -March; grey line), following the deployment of the clinical reminder (May -July; black line), and after the virtual clinic intervention (August -November; dashed line).CLL = chronic lymphocytic leukemia; PCV = pneumococcal conjugate vaccine. ", "section_name": "DISCUSSION", "section_num": null } ]	[ { "section_content": "This work was support by an educational grant from Pfizer (RJ, FP).This work was also supported in part by funds from the Cleveland Geriatric Research Education and Clinical Centers (GRECC) (FP, RB, BW, RJ) and from the National Institutes of Health (NIH), through the Clinical and Translational Science Collaborative of Cleveland (UL1TR000439) from the National Center for Advancing Translational Sciences (NCATS) component of the NIH and NIH Roadmap for Medical Research (FP, RJ).The content is solely the responsibility of the authors and does not necessarily represent the official views of the U.S. Department of Veterans Affairs, the National Institutes of Health, or the United States Government. FP and RJ designed and implemented the virtual clinic and also provided oversight for the project.RB, BW, and EC reviewed charts and analyzed data.EC wrote the initial manuscript draft, on which all authors subsequently commented. ", "section_name": "ACKNOWLEDGMENTS", "section_num": null }, { "section_content": "We have read and understood Current Oncology's policy on disclosing conflicts of interest, and we declare that we have none. ", "section_name": "CONFLICT OF INTEREST DISCLOSURES", "section_num": null }, { "section_content": "", "section_name": "AUTHOR AFFILIATIONS", "section_num": null } ]
10.1038/bcj.2015.14	Genetic and epigenetic profiling of CLL disease progression reveals limited somatic evolution and suggests a relationship to memory-cell development	<jats:title>Abstract</jats:title><jats:p>We examined genetic and epigenetic changes that occur during disease progression from indolent to aggressive forms of chronic lymphocytic leukemia (CLL) using serial samples from 27 patients. Analysis of DNA mutations grouped the leukemia cases into three categories: evolving (26%), expanding (26%) and static (47%). Thus, approximately three-quarters of the CLL cases had little to no genetic subclonal evolution. However, we identified significant recurrent DNA methylation changes during progression at 4752 CpGs enriched for regions near Polycomb 2 repressive complex (PRC2) targets. Progression-associated CpGs near the PRC2 targets undergo methylation changes in the same direction during disease progression as during normal development from naive to memory B cells. Our study shows that CLL progression does not typically occur via subclonal evolution, but that certain CpG sites undergo recurrent methylation changes. Our results suggest CLL progression may involve developmental processes shared in common with the generation of normal memory B cells.</jats:p>	[ { "section_content": "A long-standing model of cancer evolution is that tumor cells progress through stages via a reiterative process of expansion, genetic diversification by somatic mutation, and positive selection of subclones containing specific mutations. 1In practice examining tumor evolution during progression from indolent to aggressive disease is challenging as multiple biopsies are required at different time points before treatment.Chronic lymphocytic leukemia (CLL) is well suited to study tumor evolution because patients are monitored closely via blood samples until symptoms necessitate treatment. 2In addition, CLL has been shown to often harbor multiple subclones, 3 which could undergo selection during disease progression. 4A recent study used high-depth exome sequencing to examine 12 CLL patients at two time points (before and after treatment) showing clonal evolution occurring in 70% of patients, 5 consistent with the results of other sequence-based studies. 3Although these studies examined fewer longitudinal CLL samples without intervening treatment, they observed that clonal evolution is less common before treatment, 3,5 which is consistent with the results of larger-scale CLL array profiling studies. 6imitations from these studies are in the documentation of disease progression and in the limited genetic loci examined.High-depth exome sequencing studies have not yet been conducted on a large number of longitudinal samples sampled both at the time of diagnosis and immediately preceding treatment, therefore the extent and nature of clonal evolution before treatment is currently unknown. Epigenetic modifications, including DNA methylation, have long been implicated in cancer and are suspected to be oncogenic. 7][10] In cancer cells, de novo methylation occurs at these regions via recruitment of DNA methyltransferases by PRC2. 8A previous longitudinal study of CLL samples using serial samples from patients either at diagnosis and after therapy or at two time-points before treatment found that global DNA methylation may be relatively stable over time. 11owever, an in-depth genome-wide analysis of the methylation changes that occur during CLL progression in patients from near diagnosis to when they require therapy has not yet been performed; it is unknown whether specific CpG sites recurrently undergo de novo methylation or whether this involves PRC2. Here we examine 27 patients who presented with early-stage CLL disease and ultimately progressed clinically to active disease requiring treatment.We characterize somatic mutation and DNA methylation changes using paired longitudinal samples taken from these patients both at the time of diagnosis and after clinical progression, but before treatment.We show that the majority of CLL cases display-limited genetic change during that time interval, but that recurrent epigenetic changes at memory B-cell-specific PRC2 targets are associated with disease progression. ", "section_name": "INTRODUCTION", "section_num": null }, { "section_content": "", "section_name": "MATERIALS AND METHODS", "section_num": null }, { "section_content": "From over 900 CLL Research Consortium participants, 27 patients were chosen based on the following criteria: (1) received treatment at UCSD; (2) had a leukemia cell sample collected within approximately a year post diagnosis; and (3) had a leukemia cell sample collected within approximately a year before treatment.Twenty-six patients did not receive treatment before collection of samples at both time points, but one patient (SU77505) received one course of high-dose methylprednisolone and rituximab, to which the patient achieved a partial response and then subsequently relapsed before collection of the second sample.The treated sample was initially included in error but retained because the patient only achieved a partial response and did not appear genetically or epigenetically different from the other pretreatment leukemias.For each patient, leukemia cell samples were collected at multiple time points and frozen viably.The tumor fraction had to be at least 80% at one time-point (leukemia cells CD5+/CD19+ positive by flow cytometry) for inclusion in the study.For 19 patients germline DNA was isolated from saliva samples collected after treatment.The UCSD IRB approved the study and all subjects gave informed consent. ", "section_name": "Sample collection", "section_num": null }, { "section_content": "Somatic copy number alterations (sCNAs) were identified in 19 of the patients by analyzing matched normal saliva and leukemia cell samples hybridized to Illumina Omni 2.5 BeadChip arrays.CNAs were predicted using the copy number prediction algorithm CNVPartition (v3.1.6;minimum probe count of 10) and verified by visual inspection.sCNAs are reported when the estimated CNV value does not equal 2 and the CNV Confidence greater than the default of 35.Additionally, sCNA regions that have been previously implicated in CLL (chr11, chr12, chr13, and chr17) were inspected visually in all samples.To obtain the number of sCNA per individual, sCNA calls near each other on the same chromosome and at multiple time points were grouped together as an sCNA group and counted as one variant.Changes between time points were assessed by visual inspection. ", "section_name": "sCNA predictions", "section_num": null }, { "section_content": "Sequencing libraries were prepared and captured using SureSelect Human All Exon 50 Mb kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer's instructions. 12Genomic DNA (2.5 μg) was sheared to ~175 base pairs and 100 bp paired-end reads were sequenced using the Illumina Hi Seq 2000 (San Diego, CA, USA) to 75-150 × depth.Reads were aligned to the human genome reference sequence (UCSC assembly hg19) using BWA 13 with seed length set to 35.Duplicate reads were removed using Picard MarkDuplicate.GATK was used to realign reads around indels 14 based on dbSNP135 and the 1000 Genomes Project indel call set.After realignment, which can create reads that have the same position as other reads, we removed the few additional duplicate reads using Picard. ", "section_name": "Exome sequencing", "section_num": null }, { "section_content": "The single-nucleotide variants (SNVs) and indels were called using GATK v2.1-9-gb90951c UnifiedGenotyper 14 with parameters -stand_call_conf 20.0, -stand_emit_conf 10.0 and -dcov 800.UCSC assembly hg19 and dbSNP137 were used as references.Variants were recalibrated using GATK Variant Quality Score Recalibration with metrics QD, HaplotypeScore, MQRankSum, ReadPosRankSum, FS and MQ and a 99% threshold.Somatic variants were identified using a 2 × 3 χ 2 -test (chisq.test in R with permutation) across germline and the two leukemia samples.Sites with a P-value o 0.001 and a germline frequencyo0.10were called somatic. ", "section_name": "Variant calling", "section_num": null }, { "section_content": "We performed deep-targeted sequencing using the Illumina TruSeq Custom Amplicon kit (San Diego, CA, USA) modified to include singlemolecule tagging to remove duplicate reads and processed as previously described. 15Read coverage for each allele in each sample was identified using the Allele Count variable in GATK.Allele frequency differences were tested across leukemia samples using a 2 × 2 Fisher's Exact test and the targeted sequencing allele counts.Sites were considered to have changed if they were significantly different between samples (Benjamini-Hochberg false discovery rate (BH-FDR)o 0.05). ", "section_name": "Identification of somatic variants with frequency changes", "section_num": null }, { "section_content": "PyClone (v.0.12.3) was downloaded (http://compbio.bccrc.ca/software/pyclone/) and used to estimate the number of clones in leukemia samples from each patient.Copy number estimates were imported from array data (CNVPartition calls).Allele counts were obtained from targeted resequencing data.Using validated somatic variants, samples from each patient were run together using default settings. ", "section_name": "PyClone", "section_num": null }, { "section_content": "We characterized changes in DNA methylation during disease progression using the Illumina HumanMethylation450 BeadChip following manufacturer instructions.Beta values were normalized and backgroundsubtracted according to manufacturer recommended practices using GenomeStudio.Because samples were compared in pairs from the same individual, we did not remove sites that overlapped single-nucleotide polymorphisms nor process type I and type II probes separately. ", "section_name": "Methylation arrays", "section_num": null }, { "section_content": "Cell mixtures can be deconvoluted using differentially methylated CpGs that are cell-type specific. 16As the leukemia cells were not sorted we computationally corrected for methylation changes by estimating the relative composition of five cell types (B, natural killer, CD4+ T, CD8+ T and Neutrophils) and adjusted the methylation levels for the cell-type composition using linear regression.We initially observed that an unadjusted composite methylation score was positively associated with change in %CD5+/CD19+ FACS counts, suggesting an increase in sample tumor proportion during progression (Supplementary Figure 1a), which was recapitulated using just the B-cell-specific sites (Supplementary Figure 1b). 17To verify that the B-cell score was associated with leukemia cell load, we sorted six samples from three patients, cells were positively sorted for CD5+/CD19+ using anti-CD19 FITC microbeads multisort kit and anti-CD5 APC microbeads (MACS Miltenyi Biotec, Auburn, CA, USA) and compared the change in B-cell scores with differences in %CD5+/CD19+ cells (Supplementary Figure 1c).We further validated the B-cell score by comparing the change in mean somatic allele frequency with the B-cell methylation score (Supplementary Figure 1d).Methylation levels that were adjusted for all five cell types were used in downstream analyses. ", "section_name": "Adjustment of methylation beta values for cellular composition", "section_num": null }, { "section_content": "Adjusted and unadjusted beta values were tested for changes during progression using a Wilcoxon Signed Rank test (wilcox.test in R).For each patient, methylation values for each site in the first and second leukemia samples were paired.Sites were considered significant at BH-FDRo 0.05. ", "section_name": "Changes in methylation", "section_num": null }, { "section_content": "Genes were tested for enrichment using GO-seq, 18 adjusting for the number of methylation probes associated with each gene. 19Sites were annotated to one or more genes based on the Illumina HumanMethyla-tion450 BeadChip manifest file.Gene categories were downloaded from the Molecular Signatures Database v4.0 (http://www.broadinstitute.org/gsea/msigdb/index.jsp,msigdb.v4.0.symbols.gmt)][22][23][24] Overlap across sites was assessed using a Fisher's Exact Test (fisher.exact in R).If previously published results were based on the 27 K array, we restricted the comparison with those CpGs in our study that were also on the 27 K array, based on Illumina Target ID. Histone modification enrichment analysis ENCODE ChIP-seq peaks (405 experiments) were downloaded from UCSC (http://hgdownload.cse.ucsc.edu) on 27 February 2013.Overlaps between significantly differentially methylated probes and ENCODE ChIP-seq peaks were computed by intersecting 200 bp regions centered on significant probes with ChIP-seq peaks using bedtools intersectBed. 25Enrichment P-values were calculated using a hypergeometric test. ", "section_name": "Gene set association", "section_num": null }, { "section_content": "", "section_name": "RESULTS", "section_num": null }, { "section_content": "We collected serial blood samples from 27 patients shortly after diagnosis and before they required treatment (median years between time points was 1.6 years; range 0.3-9.9years).Our cohort included patients with both low-and high-risk prognostic factors (Figure 1).All patients presented at diagnosis with earlystage disease that progressed over time to disease requiring therapy by the International Workshop on CLL (iwCLL) criteria. 2e identified somatic copy number alterations (sCNAs), detecting 30 sCNAs in 19 patients corresponding to an average of 1.7 sCNAs genome-wide per patient (range, 0-6; Figure 2a and Supplementary Table 1).sCNAs previously implicated in CLL were common with 13/19 (68%) of patients showing an sCNA in at least 1 of 4 recurrently altered loci (Figure 2b and Supplementary Table 1).The majority of sCNAs were observed at both sample collection time points.However, we observed two instances of 11q deletions and one instance of a novel sCNA (SU13717) that were only present in the latter sample (Figure 2b and Supplementary Table 1).These results are in agreement with studies showing that over 80% of CLL cases have sCNA that are frequently subclonal. 26e identified somatic point mutations through whole-exome sequencing of matched germline and leukemia cell samples at both clinical time points.In total, we identified 871 somatic SNVs (sSNVs) corresponding to 26-80 coding or splice site point mutations per patient (Figure 2a, Supplementary Table 2).We identified indels in three genes (to increase confidence indel analysis was restricted to known recurrently mutated genes in CLL; Supplementary Table 2).We validated the somatic mutation calls by deep-targeted sequencing (~850 × coverage) using the Illumina TruSeq Custom Amplicon (TSCA) kit (San Diego, CA, USA) modified to account for PCR duplicates. 15We interrogated 521/871 (60%) of the sSNVs and validated 484/521 (93%) of these sites (Supplementary Table 3).The vast majority of validated sSNVs were observed at both time-points with only one leukemia case displaying mutations unique to the second time-point-a deletion in NOTCH1 and an sSNV in DDX3X.Although 13/871 of the sSNVs we observed were in recurrently mutated genes (Figure 2b, Supplementary Table 2), the vast majority were novel and likely passenger mutations. 3 examine subclonal patterns of change during tumor progression, we analyzed the 484 validated sSNVs (mean allelic fraction of 39% (range 0-97%) at the first time point) for significant changes in allele frequency during disease progression.Overall, 109/484 (23%) of the sites changed significantly (FDRo 0.05), of which only 37/109 (34%) had a 410% allele frequency change (Figure 2c and Supplementary Table 3).Because the CLL cells were not sorted, some changes in allele frequency during progression could be due to the leukemia expanding relative to the normal mononuclear cells.To differentiate this from clonal evolution where one subclone is increasing in frequency in comparison to other subclone(s), we three different categories: (1) evolving, where at least one sSNV increased and at least one decreased in frequency (N = 5); (2) expanding, where two or more sSNVs increased in frequency and none decreased (N = 5); and (3) static, where no or only one sSNV increased or decreased in frequency (N = 9; Figure 2c).Evolving cases had an average median allele frequency change of ~9% (range 6-14%) and on average ~44% (range 17-88%) sSNVs changed frequency.Expanding cases had an average median allele frequency change of ~7% (range 5-9%) and on average ~28% (range 18-42%) sSNVs that changed frequency (Supplementary Table 4).Thus the absolute changes in sSNV frequencies across the two-sample collection time points were comparable between the evolving and expanding leukemia cell populations, but in the former particular subclones were expanding and others contracting, whereas in the latter the leukemia was likely expanding relative to the normal population.These data show that in ~25% of CLL cases, subclonal somatic evolution does occur with new sCNAs and point mutations occurring or significantly changing in frequency, but in most cases, mutations are present at the time of diagnosis and show limited change in their relative frequencies. CLL somatic evolution category and prognostic risk factors Although we are limited by sample size, we explored if significant relationships exist between the somatic evolution categories and prognostic factors, time to progression, presence of driver mutations or extent of intraclonal genetic heterogeneity.Patients with CLL cells that use unmutated immunoglobulin heavy chain (IGH) variable region (IGHV) and/or express high ZAP-70 generally have a more aggressive clinical course; 27 however, such prognostic factors were not associated with a particular category of CLL (Figures 2d ande).Time to treatment was also not associated (Figure 2f).We also do not observe a strong association with the presence of somatic mutations in nine genes previously shown to be recurrent in CLL and/or high-risk sCNAs with any one of the three different categories (Supplementary Table 2, Figure 2b).However, we observed a nominal association between the 11q deletion and the evolving category compared to the combined group of expanding and static leukemias (P = 0.04, unadjusted P-value).Only two mutations in driver genes significantly changed allele frequencies, both in the same individual (SU35420) (Figure 2b).We additionally examined the relationship between CLL category and intraclonal heterogeneity estimated using PyClone. 28We observed a total of 90 subclones with each leukemia case carrying an average of 5 (range 2-9) subclones (Figure 2g).However, we observed no statistically significant difference between evolving leukemias vs. expanding and static leukemias combined (Mann-Whitney P = 0.4).These results suggest that some somatic variants, such as 11q deletions, may be associated with somatic evolution category, but larger sample sizes will be required to define associations that are statistically significant. ", "section_name": "CLL cells typically display-limited genetic change during disease progression", "section_num": null }, { "section_content": "We profiled the leukemia cell samples from 27 patients at both clinical time points using Illumina HumanMethylation450 Bead-Chip arrays (450k arrays).After adjusting methylation levels for cellular composition (see Methods), we identified 4,752 CpG sites co-located near 2,670 genes that showed significant changes in the leukemia cells during progression (Supplementary Table 5).We did not observe clustering of methylation patterns at these 4,752 CpGs sites associated with time to progression or somatic evolution category.Instead, they appeared heterogeneous with each individual having a different combination of sites affected (Figure 3a).Considering all patients, the majority of sites (3670/ 4752, 77%) showed modest increases in methylation (median increase of 2.9%, range 0.5-9.5%).Changes for a given patient were also modest, with 49 sites on average changing more than 20% and 1220 changing more than 5% (Figure 3a).The leukemias with the least amount of methylation changes were of the static evolution category and from patients that had progressed relatively quickly after diagnosis (Figures 1 and3a).However, there were instances of static disease with longer times between diagnosis and treatment that had high numbers of changes, indicating that methylation changes do not require genetic subclonal evolution.Although leukemias with the highest numbers of CpG sites that changed were in the expanding or evolving categories, there were leukemias in each of the three categories that had relatively low numbers of methylation changes.These results indicate that methylation changes occurring at the 4752 progression-associated CpGs are modest in effect size, heterogeneous across individuals and are not specific to a certain somatic evolution category. ", "section_name": "CLL cases show methylation changes at specific CpG sites during disease progression", "section_num": null }, { "section_content": "To assess the biological relevance of the methylation changes at the 4752 progression-associated CpGs, we performed gene set analysis using a method that corrects for the biased representation of probes to genes on the 450k arrays. 19We observed significant differences between CpG sites that increase or decrease in methylation (Figure 3a) during CLL progression; those that increase are strongly enriched near genes associated with H3K27me3 modifications and PRC2 targets (Supplementary Table 6), whereas those that decrease show no significant relationship.Further analysis showed that the CpGs most commonly experiencing methylation changes were significantly clustered around genes with H3K27me3-marked promoters in naive B-cells (P = 1 × 10 -33 ), germinal center B-cells (P = 9 × 10 -21 ) and embryonic stem cells (P = 3 × 10 -41 ; Supplementary Table 6).They also were clustered around genes bound by EZH2 in germinal center B-cells (P = 3 × 10 -29 ) and hESCs (P = 1 × 10 -23 ). 20,29These results show that the PRC2 is bound normally in B-cells near CpGs that undergo hypermethylation during disease progression in patients with CLL.We further explored colocalization between the 4572 progression-associated CpGs and chromatin modifications within ± 100 bp of ChIP-seq peak using 406 experiments by the ENCODE project. 30We observed strong enrichment of regions marked by H3K27me3 and H3K9me3, and bound by PRC2 components SUZ12 and EZH2 (Figure 3b). We compared our findings with those of previous methylation studies of CLL and aging.The promoters of several genes known to be methylated in CLL samples (SFRP1, SFRP2, DAPK1, CDH1) had CpG sites that were significantly associated with disease progression (Supplementary Table 5). 31,32We did not, however, observe changes in methylation in ZAP70, indicating that this prognostic marker does not apparently change over time. 33We examined the overlap with genes associated with CpG sites that show changes in the CLL mouse model of TCL1A overexpression and observed a significant overlap with 23/51 genes that showed changes in this mouse model near a progression-associated site (P = 0.0002) identified in our study. 213][24] We observed little overlap with 353 CpGs that show age-acceleration in cancer tissues (4/353 significant, Fisher P = 0.56) or 96 CpGs found to be associated with age in blood (2/96 markers, Fisher P = 0.24). 22,24e did, however, observe an overlap between progressionassociated sites and age-associated sites that are enriched for Polycomb target promoters (8/207 markers, Fisher P = 0.001). 23hese data demonstrate that the CpGs associated with CLL progression frequently overlap genes previously shown to be differentially methylated in CLL, and that changes in methylation status are unlikely to be solely due to increased cellular age or cellular composition changes associated with aging. Progression-associated CpGs show methylation changes similar to those in normal memory B-cell development We compared the methylation levels at the 4752 progression CpGs of the 27 paired leukemia samples in our study to that of six naive (CD5+ and normal) and six memory B cells (class switched and nonclass switched) using 450 k array data from the International Cancer Genome Consortium (ICGC). 34Using hierarchical clustering, we observed that in all patients the methylation status at these CpG loci were more similar to that of memory B cells than to that of naive B cells (Figure 4a).We then examined whether methylation changes at the 4752 progression-associated CpGs were similar to changes that happen during leukemia initiation and/or normal B-cell development.To do this, we identified CpGs that differed by at least ± 10% between naive, memory and the first CLL time point and identified those that overlapped with progression CpGs (Figure 4b).Considerably more CpGs change methylation status between naive and memory B-cells or between naive B cells and the first CLL samples then between memory B cells and the first CLL time point.However, we observed a significant enrichment for progression CpGs in total (and even more so for those near H3K27me3) with sites that differed between pair-wise comparisons of all three cell types (naive B cells, memory B cells and the first CLL sample) as compared with all CpGs (Figure 4b).For progression CpGs near H3K27me3, we observed a strong overlap between the sites that differed between naive and memory and those that differed between naïve and the first CLL sample (Figure 4d).For these overlapping sites, methylation changes during progression and differences between naive and memory and naive and CLL first Sites that showed significant differences were tested for whether they were likely to be near regions bound by modified histones or bound by the EZH2 and SUZ12 proteins as measured by ChiP-seq in a variety of cell types using the ENCODE data.Significant enrichment is indicated with a red bar.If no mark is present, the assay was not performed. time point were largely in the same direction (Figure 4e), particularly for CpGs near H3K27me3 that become more methylated during progression.These data show that the majority of CpGs that undergo methylation change during CLL disease progression undergo changes in the same direction during normal memory B-cell development. Previous work suggested that CLL with mutated or unmutated IGHV are derived from memory and naive B cells, respectively.However, we did not observe an overlap between 3265 CpGs associated with IGHV mutation status and the 4752 progressionassociated (P = 0.64). 34These findings suggest that although the CpGs that undergo methylation changes during progression overlap those that change during the development from naive to memory cells, or transition from naive to CLL, they are not the same as those that differ between IGHV-mutated and unmutated CLL or those that are change during the transition from memory cells to CLL. As the leukemia cell samples from the 27 patients in our study were not sorted we adjusted methylation levels for cellular composition computationally; therefore, we performed additional analyses using sorted cells to validate our findings.To examine changes during progression, we positively sorted serial samples from three patients for CD5+/CD19+ cells and performed 450 k arrays.We averaged the change in methylation during progression and identified sites that changed the most (top and bottom 5% quantiles).We compared these with the progressionassociated sites and we observed positive enrichment (OR = 1.35,P = 1.1 × 10 -11 ), showing that progression-associated CpGs are likely to show changes in sorted CLL cells during progression.In addition, sites in the top 5% quantile were enriched for sites near H3K27me3 regions (OR = 1.79,P = 3.7 × 10 -238 ).To test whether sorted CLL cells look more similar to memory B cells at progression-associated sites, we examined the methylation levels of the 139 sorted CLL cells in the ICGC dataset relative to naive and memory B cells (Figure 4f).We observed clustering within CLL according to IGVH mutation status; however, both types of CLL were more similar to memory B cells than naive at these 4752 sites.In addition, progression-associated sites near H3K27me3 regions predominantly showed higher methylation in the CLL and memory B cells compared with naive.These results provide an important control and suggest that our findings based on data from compositionally corrected CLL samples are valid in sorted CLL cells. ", "section_name": "CpGs associated with CLL progression are near PRC2 target genes in B cells", "section_num": null }, { "section_content": "This study provides insight into the genetic and epigenetic changes that occur during progression from an indolent form of cancer to a physiologically more aggressive form.Our findings are inconsistent with the dogma of cancer progression through stages via somatic mutation followed by positive selection of subclones containing specific mutations. 1In contrast to evolution documented in pre-vs post-treated samples, 3 we observed little clonal evolution during progression from indolent to aggressive disease, suggesting that changes observed in treated samples may arise during repopulation of the cancer and are not required for the leukemia to become more aggressive.Many of the tumors that showed static somatic evolution and low methylation score changes progressed to require treatment quickly (o2 years).It is possible that these tumors were aggressive at the point of diagnosis and showed no or undetectable further genetic and epigenetic changes during progression.Because these patients progressed clinically between the sample time points, however, this suggests that clinical progression can occur with little genetic or epigenetic change. We observed coincident changes in DNA methylation associated with Polycomb repression across the majority of CLL cases.PRC2 target methylation in cancer is common; 8 however, the methylation patterns observed are usually similar to those in stem cells. 35The changes that we observe occur more often at sites that undergo epigenetic modifications during normal differentiation of naive to memory B cells.This suggests that clinical progression from indolent to active forms of CLL may involve developmental processes shared in common with those involved in normal B-cell differentiation. In the 26% of leukemia cases that do show clonal evolution, there may be somatic drivers involved, such as NOTCH1 or chr11 deletions (including the interval encoding ATM), as has been suggested by previous work. 36We observed three instances of 11q deletions occurring in leukemias that showed clonal evolution, with two instances arising during progression, findings consistent with a late-acting role for ATM.Both ATM and TP53 are DNA damage response genes recurrently mutated or deleted in CLL and associated with poor prognosis.We hypothesize that effects of ATM and TP53 mutations may be through the PRC2.ATM-mediated phosphorylation of EZH2 has been shown to reduce protein stability, reducing PRC3 formation and increasing H3K27me3 in cells with ATM deficiency. 37TP53, which is downstream of ATM, has also been linked to H3K27me3 through one of its targets, the lincRNA LINC-PINT. 38These new roles for ATM and TP53 that are unrelated to their DNA damage response functions may help to explain how mutations in these genes are advantageous for CLL cells despite the very low rates of somatic mutation in CLL and little evidence of clonal selection pre-treatment. In summary, we have traced the molecular changes at genetic and epigenetic levels in CLL cases as they clinically progress from indolent to active disease requiring therapy.We show that CLL progression does not typically occur via genetic clonal evolution, but that that certain CpG sites undergo methylation consistent with an increase in PRC2 activity.Our results suggest that changes in epigenetic regulation via the PRC2 occur during CLL progression and may involve developmental processes shared in common with the generation of normal memory B cells.Interestingly, memory B cells share hematopoietic stem cell features that may be advantageous to CLL, including the ability to undergo longterm self-renewal. 39Further research into the role of PRC2 and how it interacts with recurrently mutated genes in CLL may provide insight into the molecular mechanisms underlying CLL disease progression. ", "section_name": "DISCUSSION", "section_num": null } ]	[ { "section_content": "We thank the Projects teams at the Ontario Institute for Cancer Research, Canada.We also thank the patients for participating in this study.This work was funded in part by the National Cancer Institute (1R21CA152613, P30CA023100), the CIRM Highly Active Anti-Leukemia Stem Cell Therapy (HALT) team grant (DR1-01430), the National Institutes of Health Grant UL1TR000100 and the Government of Canada through Genome Canada and through the Canadian Institutes of Health Research (CSC-105367).The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.PJS was supported by the UCSD Cancer Center Training Grant in Drug Development-the Cancer Therapeutics Training Program T32CA121938.This work was also supported by the Cancer Stem Cell Consortium with funding from the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-047), and through the Canadian Institutes of Health Research (CSC-105367).TJH and LM received Investigator Awards from the Ontario Institute for Cancer Research, through generous support from the Ontario Ministry of Research and Innovation.Copy number arrays, exome sequencing data, targeted sequencing data, and methylation array raw data will be available through dbGaP accession phs000767: http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000767.v1.p1. ", "section_name": "ACKNOWLEDGEMENTS", "section_num": null }, { "section_content": "KLG is an employee of Illumina and may own stock in the company.The remaining authors declare no conflict of interest. ENS, CMD, KY, PJS, MB and HM performed the analyses.ENS, CMD, EMG and KAF wrote the manuscript; EMG, LZR and TJK coordinated clinical information and provided samples.KJ and OH oversaw laboratory experiments.LM and TH contributed copy number array data sets.LZR, EMG, YD, MK, SR and HA performed laboratory experiments.KLG collected HumanMethylation450 data.HM and CLB provided bioinformatics support.ENS, DAC, CHMJ, KM, TJK and KAF designed the study.KAF and TJK oversaw the work.Supplementary Information accompanies this paper on Blood Cancer Journal website (http://www.nature.com/bcj) ", "section_name": "CONFLICT OF INTEREST", "section_num": null }, { "section_content": "KLG is an employee of Illumina and may own stock in the company.The remaining authors declare no conflict of interest. ", "section_name": "CONFLICT OF INTEREST", "section_num": null }, { "section_content": "ENS, CMD, KY, PJS, MB and HM performed the analyses.ENS, CMD, EMG and KAF wrote the manuscript; EMG, LZR and TJK coordinated clinical information and provided samples.KJ and OH oversaw laboratory experiments.LM and TH contributed copy number array data sets.LZR, EMG, YD, MK, SR and HA performed laboratory experiments.KLG collected HumanMethylation450 data.HM and CLB provided bioinformatics support.ENS, DAC, CHMJ, KM, TJK and KAF designed the study.KAF and TJK oversaw the work.Supplementary Information accompanies this paper on Blood Cancer Journal website (http://www.nature.com/bcj) ", "section_name": "AUTHOR CONTRIBUTIONS", "section_num": null } ]
10.3390/jof7030212	Cryptococcus gattii in Patients with Lymphoid Neoplasms: An Illustration of Evolutive Host–Fungus Interactions	<jats:p>Recent outbreaks of Cryptococcus gattii (CG) infections in North America have sparked renewed interest in the pathogenic potential of CG, and have underscored notable differences with Cryptococcus neoformans in terms of geographic distribution, pathogen virulence, and host susceptibility. While cases of CG are increasingly reported in patients with a wide variety of underlying conditions, only very few have been reported in patients with lymphoid neoplasms. Herein, we report a case of autochthonous CG meningitis in a patient receiving ibrutinib for chronic lymphocytic leukemia in France, and review available data on the clinical epidemiology of CG infections in patients with lymphoid neoplasms. We also summarise recent data on the host responses to CG infection, as well as the potential management pitfalls associated with its treatment in the haematological setting. The clinical epidemiology, clinical presentation, and course of disease during infections caused by CG involve complex interactions between environmental exposure to CG, infecting genotype, pathogen virulence factors, host susceptibility, and host immune responses. Future treatment guidelines should address the challenges associated with the management of antifungal treatments in the onco-haematological setting and the potential drug-drug interactions.</jats:p>	[ { "section_content": "Cryptococcosis is one of the most frequent causes of invasive fungal infections causing human disease, and the most common cause of meningitis in adults living with HIV in Sub-Saharan Africa [1][2][3].Cryptococcosis is caused by Cryptococcus neoformans (CN) and Cryptococcus gattii (CG).While CN has by far been the focus of most research due to its importance as an AIDS-defining pathogen, the clinical epidemiology of cryptococcosis caused by CG is relatively less defined [4].Recent outbreaks of CG infections in North America, primarily caused by a virulent strain of the VGII lineage, have sparked renewed interest in the pathogenic potential of CG and have underscored notable differences with CN in terms of geographic distribution, pathogen virulence, and host susceptibility [5].Cases of both CN and CG have been reported in patients with haematological malignancy [6,7].In particular, there have been sporadic reports of cases of cryptococcosis in patients receiving ibrutinib for lymphoid neoplasms.Herein, we report a case of meningitis caused by CG in a patient receiving ibrutinib for relapsed chronic lymphocytic leukaemia, and review the clinical epidemiology of CG infections during haematological malignancy.We also summarise recent data on the host responses to infections caused by CG and its management in the setting of haematological malignancy. ", "section_name": "Introduction", "section_num": "1." }, { "section_content": "An 88-year-old woman was referred to Necker-Enfants Malades Hospital in Paris, with a 5-day history of fever and headache.The patient had a history of relapsed chronic lymphocytic leukaemia (CLL), for which she had received successive treatment lines of chloraminophene, rituximab, chloraminophene, and lastly, rituximab and bendamustine.Due to relapse, the patient was currently receiving ibrutinib (420 mg/day), which had been started 8 months prior to admission.The patient resided in a house located in the south-east of France.She had no history of travel but reported extensive exposure to eucalyptus trees around and inside her house.At admission, we noted psychomotor slowing and meningeal syndrome.Blood counts showed neutropenia (0.5 × 10 9 cells/L, reference range 4-10 × 10 9 cells/L) and lymphopenia (1.4 × 10 9 cells/L, reference range 1.5-4 × 10 9 cells/L).C-reactive protein level was at 68 mg/L (reference < 5 mg/L) and CD4+ lymphocyte counts were at 186/µL (reference > 700/µL).Serum gamma globulins were markedly reduced with IgG levels at 0.99 g/L (reference range 6.6-12.8g/L).Analysis of cerebrospinal fluid (CSF) showed pleocytosis with 116 cells/mL (96% lymphocytes), elevated protein levels (0.7 g/L, reference < 0.4 g/L), and hypoglycorrhachia (0.33 g/L, reference > 0.5 g/L).Theopening pressure of CSF was elevated (25 cm H 2 O, reference < 20 cm H 2 O).An India ink test on CSF was positive.Cryptococcus antigen (CALAS, Meridian Bioscience) was positive in the CSF (titre of 11) and in the serum (titre of 19).Fungal cultures of the CSF were positive and the MALDI-TOF method (Bruker Biotyper) allowed identification of the species Cryptococcus gattii.Genotyping was performed by multilocus sequence typing (MLST) at the French National Reference Center for Invasive Mycoses and Antifungals (NRCMA), using the ISHAM-7 loci scheme and comparisons with the online database (http://mlst.mycologylab.org/page/CG%20,main accessed on 22 October 2020) [8], which led to the identification of Cryptococcus gattii serotype B, belonging to the molecular clade VGI, genotype ST197.This genotype belongs to the European-Mediterranean cluster identified by Hagen et al. [9].Mycological blood cultures were negative, and cerebral magnetic resonance imaging was unremarkable.Chest computed tomography images showed a 2 cm × 3 cm mass lesion of the upper left lobe, evocative of a CG cryptococcoma.Testing for anti-granulocyte-macrophage colony stimulating factor (GM-CSF) autoantibodies was negative.We administered intravenous liposomal amphotericin B (L-AMB) (3 mg/kg/day) and 5-fluorocytosine (5-FC) (100 mg/kg/day), and ibrutinib was suspended.After 14 days of combination induction therapy, neutropenia worsened (0.2 × 109 cells/L) and antifungal treatment was switched to oral fluconazole (800 mg/day).Four weeks after treatment initiation, neutrophil counts were within normal ranges (2.2 × 109 cells/L).At week 12, ibrutinib was reintroduced due to lymph node growth, albeit at a reduced dosage (140 mg/day) because of concerns regarding drug-drug interactions between fluconazole and ibrutinib.Repeated cryptococcal antigen assays at 3 months of treatment initiation showed marked reduction in titres (4 and 3.1 in the CSF and serum, respectively).The patient is currently still receiving ibrutinib, and has experienced no relapse of infection or CLL after more than two years of follow-up. ", "section_name": "Case Description", "section_num": "2." }, { "section_content": "CG is an encapsulated basidiomycetous yeast from the Cryptococcus species complex, which grows preferentially in over 50 species of trees, including eucalyptus trees, and in their hollows [10,11].The CG species comprises two serotypes, B and C, and can be further subdivided into five molecular lineages (VGI-V) [12], with VGV having only recently been identified in the Central Miombo Woodlands of Zambia, Africa [13].Identification of CG at a molecular subtype level appears to be important, as these lineages seem to differ in geographic range, host susceptibility, and antifungal susceptibility [5,14,15].Cases of endemic or sporadic CG infections have been reported for all molecular lineages, in a wide range of regions, including Australia and Papua New Guinea (VGI and VGII), South America-including French Guiana-(VGI-IV), Southeast Asia (VGI-IV), Southern and Central Africa (VGIV and VGV), and the United States (VGI and VGIII), as well as more recently in an outbreak setting in North America (VGIIa and VGIIb) [10,13,[16][17][18].In Europe, there have also been sporadic reports of CG, a majority of which are believed to be imported or travel-related, although autochthonous cases have been reported as well [9].Indeed, there is increasing evidence that CG has been emerging in Europe over the past two decades [9,19], and a Mediterranean cluster belonging to genotype VGI was found in environmental and clinical isolates recovered from southern Italy, Portugal, and France [9,20]. Precise data on the incidence and clinical presentation of CG are biased by the absence of routine identification at a species level in most cases of cryptococcosis outside of endemic and outbreak settings [5,14].Nevertheless, prior to the description of outbreak cases in North America in the early 2000s, the clinical epidemiology of CG was thought to be distinct from that of CN.Historically, infections by CG were believed to be geographically limited to tropical or sub-tropical regions [16].In addition, whereas only 10-20% of patients with CN infections were seemingly immunocompetent, this was the case for up to 80-100% of patients with endemic CG infections [21,22].Finally, CG more frequently caused focal pulmonary disease, cerebral cryptococcomas, hydrocephalus, and was more often responsible for neurological sequelae [21][22][23].This clinical paradigm has since shifted with studies of outbreak cases that started in 1999 on Vancouver Island, British Columbia, Canada, and subsequently spread to the U.S. Pacific Northwest [24,25].In these outbreak cases, 97% of which were caused by yeasts of the VGII lineage (primarily by VGIIa, and to a lesser extent by VGIIb), 38-59% of affected patients had an underlying condition (most frequently an underlying pulmonary disease or a history of systemic corticosteroid use) [26,27].Compared to CG cases reported in other locations, as well as sporadic strains from North America (i.e., of molecular type VGI), VGII outbreak strains more often presented with pulmonary involvements and were associated with unusually high case fatality rates (8-33%) [26, 28,29].Nevertheless, although the infecting genotype seems to contribute to the phenotype of the infection, it now seems increasingly likely that clinical presentation is dictated by a more complex combination of factors that also includes host immune status and environmental exposure to CG [22,30]. ", "section_name": "Clinical Epidemiology of Cryptococcus gattii", "section_num": "3." }, { "section_content": "Of the more than 30 species in the Cryptococcus genus, only CN and CG are known to be pathogenic to humans [1].While cryptococcosis caused by CN generally results from the reactivation of latent infection, almost exclusively in the setting of immunosuppression, CG is considered a primary pathogen [5].Infection occurs via inhalation of yeast cells or basidiospores from the environment, with supposed subsequent survival in the lungs within intra-alveolar macrophages.Dissemination to the central nervous system can ensue, either via transcellular migration across the blood-brain barrier, or alternatively via a paracellular passage using a 'Trojan Horse' method, during which monocytes are hijacked by Cryptococcus spp.[31]. While the specific interactions between CG and the host immune system have not been studied to the same extent as for CN, many of these seem to be similar between the two species.They involve specific virulence factors, pathogen escape mechanisms, and alterations to innate and adaptive immunity [32,33].The most studied of these virulence factors is the presence of a polysaccharide capsule, which provides evasion to immune recognition, decreases complement activity, and downregulates the host production of TNF-alpha and IFN-gamma [32].Survival of the yeast during phagocytosis is further facilitated by a mechanism of capsule enlargement, as well as the ability to escape phagocyting macrophages through exocytosis [34,35].Additional virulence factors include the antioxidant properties of melanin, which is present in the cell wall and protects the yeast from free radicals, and the ability to survive at body temperature [33].In mice models of CN infection, depletion of specific cytokines has shown that lack of IL-12 and IFNgamma production was associated with increased mortality, whereas downregulation of IL-13 and IL-4 promoted survival [35].The fact that cases of cryptococcosis have been reported in patients with circulating antibodies against IFN-gamma and in those with IL12Rβ1-deficiency seems to support the crucial role of the IL-12/IFN-gamma axis in anticryptococcal immunity [36,37].TNF-alpha appears to play an equally pivotal role in host responses to CN, illustrated by the observation that TNF-alpha-deficient mice displayed reduced survival to disseminated CN infections despite increased IL-12 and IFN-gamma levels [38].Interestingly, the cytokine pattern involved in the host response to CG was shown to differ from that of CN.In vitro studies using heat-killed isolates incubated with human peripheral blood mononuclear cells have shown that CG infection, compared to CN infection, was associated with higher concentrations of the pro-inflammatory cytokines IL-1β, TNFα, and IL-6 [39].This could help explain the severity of CNS disease oftentimes reported during non-outbreak CG infections in immunocompetent patients [40], which is characterised by high rates of hydrocephalus and immune reconstitution inflammatory syndrome (IRIS)-like manifestations [6].It has also led to the suggestion that a substantial subset of apparently immunocompetent patients with CG infections could in fact have subtle immune defects.Evidence supporting this hypothesis can be found in reports of CG infections revealing subclinical antibody deficiencies [41], as well as the discovery of circulating auto-antibodies against GM-CSF in otherwise immunocompetent patients with CG meningitis [42]. Finally, as previously mentioned, one of the major differences between CG and CN appears to be the extent to which pathogenicity and clinical presentation of CG seem to be linked to infecting genotype.Reports of non-outbreak VGI strains were primarily associated with CNS disease in immunocompetent hosts, whereas outbreak strains (VGIIa and VGIIb) were more often involved with the lungs of patients with frequent underlying conditions.Evidence for increased virulence of outbreak strains was provided in mice models, in which some outbreak genotypes (notably VGIIa) were shown to be more virulent than others in vitro [43].Interestingly, this did not simply appear to be linked to over-expression of individual pathogenicity factors in outbreak strains, but rather to an enhanced ability to proliferate in host macrophages [44].This could perhaps help explain the high rates of lung cryptococcoma found in outbreak settings compared to reports from endemic cases. ", "section_name": "Host Defences against Cryptococcus gattii", "section_num": "4." }, { "section_content": "Haematological malignancy is a recognised predisposing factor for Cryptococcus neoformans infections, albeit to a lesser extent than solid-organ transplantation and receipt of systemic glucocorticosteroids [45].Patients with lymphoid neoplasms appear to represent an emerging group of at-risk patients for cryptococccosis among those with haematological malignancy, which is comparable to what has been observed for invasive pulmonary aspergillosis in this population over the last decades [46][47][48].Although haematological malignancy appears to be the most at-risk type of cancer for cryptococcosis [7], it only accounted for under 10% of 306 cases in HIV-negative patients [45].There are only a few data on CG infections in the onco-haematological setting.In Australia, where CG is endemic, cases have been reported in patients with an increasingly wide range of immunocompromised states, with 8.5-27% having a non-HIV immunosuppressive condition [6,22].However, only four cases of non-outbreak CG infections in patients with haematological malignancy have been specifically reported, to our knowledge [6].With the British Columbia and U.S. Pacific Northwest outbreaks, new risk factors for CG infection were identified.Among 218 cases of CG infections in British Columbia from 1999-2007, 38 had a history of invasive cancer, including 13 patients with leukaemia or lymphoma [27].Patients infected with outbreak strains in the Pacific Northwest were also more likely to have a pre-existing condition.Oral corticosteroid use was the most frequent condition associated with infections by these outbreak strains (accounting for 55% of cases), and receipt of corticosteroids within the preceding year was associated with risk of death.Among specific underlying conditions, only history of cancer was statistically more often associated with outbreak strains compared to non-outbreak strains [29]. We found no reported cases of CG in the literature specifically mentioning ibrutinib receipt in the treatment of lymphoid neoplasms.This is noteworthy, considering recent reports regarding the increased risk of invasive fungal infections in patients receiving ibrutinib, an inhibitor of the Bruton tyrosine kinase [49].CLL is a malignancy of mature B cells, which exposes affected patients to an increased susceptibility to bacterial, mycobacterial, and fungal infections [50,51].This is presumably related to both disease-and treatment-related factors.CLL, including in treatment-naïve patients, is characterised by quantitative and qualitative defects in B cells, T cells, NK cells, neutrophils, monocytes, and macrophages [52].The overall incidence of invasive fungal infections remains low in CLL (estimated to range from 0.5 to 7.8% [53]), and susceptibility to cryptococcal disease in patients with B-cell malignancies seems to be particularly linked to advanced disease, pre-treatment, and longer duration of the underlying malignancy [45].Patients receiving chemotherapy regimens containing fludarabine as well as the anti-CD52 monoclonal antibody alemtuzumab appear to be at particular risk of cryptococcal disease, presumably due to further quantitative and qualitative impairments in T cell responses [54,55].More recently, there has been a particular focus on the epidemiological association between treatment regimens containing ibrutinib and the emergence of invasive fungal infections [49,56].While the majority of these reported cases concerned invasive aspergillosis, Cryptococcus neoformans infections have also sporadically been reported in patients with ibrutinib-treated lymphoid neoplasms [56][57][58][59][60][61].In 11 reported cases of CN cryptococcosis in patients with ibrutinib-treated CLL, 10/11 patients were male, and the median age was 67 years.Cryptococcosis occurred after a median of 15 (range 3-35) weeks after ibrutinib introduction, and 8/11 had prior or concurrent CLL treatments.The lungs were the most frequently affected organ (8/11), followed by the CNS (4/11) [49,[58][59][60]62,63].Susceptibility to infection in these ibrutinib-treated patients has been linked to altered B-cell receptor signaling and the inhibition of interleukin-2-inducible kinases [64], as well as impairments of innate immunity [65], most notably in neutrophil and monocyte functionality [66,67].However, the fact that a significant number of reported cases of invasive fungal disease occurred in patients with relapsed/refractory diseases may point towards a combination of factors including disease and pretreatment, rather than being caused by ibrutinib alone [57]. Interestingly, it has been suggested that the CSF inflammatory response in cases of cryptococcal meningitis could be milder in patients with haematological malignancy [34].Although this was not true in our reported patient, it was apparent in four previously reported cases of CN meningitis in patients receiving ibrutinib for whom clinical data were available (median CSF nucleated cell counts: 2/dL, range: 1-4/dL) [58,60].This was also previously reported in cases of bacterial and fungal meningitis in patients with cancer [68], and is reminiscent of the non-inflammatory CSF profile sometimes found in cases of CN meningitis in profoundly CD4-depleted HIV/AIDS patients [69].This particular aspect certainly deserves further investigation, with an emphasis on CG meningitis, which is characterised by significant CSF inflammation, and higher rates of hydrocephalus and neurological sequalae than CN [6]. ", "section_name": "Cryptococcus gattii Infections during Lymphoid Neoplasms", "section_num": "5." }, { "section_content": "Treatment guidelines for CG meningitis and meningoencephalitis are based on case series and expert opinions rather than controlled studies [5,70], but there nevertheless appear to be significant differences when compared to the management strategies for CN.Induction therapy regimens including a combination of amphotericin B (or L-AMB) and 5-FC for a duration of 6 weeks were associated with the highest success rates in cases of meningitis (although 2 weeks may be sufficient in cases of isolated pulmonary disease) [70].Up to 18 months of maintenance treatment regimens with fluconazole are required in cases of CNS disease, which is longer than that recommended for CN meningitis [5].Management of intracranial pressure is also fundamental, and often requires repeated lumbar punctures or the use of lumbar drains/CSF shunts [40].Corticosteroid use can also be necessary in light of the high rates of IRIS-like manifestations, which can occur after 1 to 12 months [70].One of the foreseeable challenges in the management of CG meningitis in the onco-haematological setting is the potential bone marrow suppression induced by 5-FC [71], which has been linked to the conversion of 5-FC to 5-fluorouracil by the intestinal microflora [72].Indeed, in patients with an underlying haematological malignancy, particularly those having undergone radiation therapy or myelosuppressive chemotherapy, this adverse effect can lead to treatment cessation.Our case illustrates this, as induction therapy had to be prematurely discontinued due to neutropenia.Drugdrug interactions between first-line antifungal agents and targeted therapies can also be of concern.Azoles in particular interfere with the elimination of ibrutinib by inhibiting the CYP3A4 enzyme system, leading to an increased risk of adverse effects [73].In all cases, the decision to continue or stop ibrutinib alongside azole-based antifungal therapy should be balanced against the risk of relapse of the underlying malignancy, especially when considering the prolonged duration of azole-based maintenance therapy.There are few data on the modalities of ibrutinib reintroduction in CLL when associated with azolebased therapies, but experts generally recommend marked dose reductions, usually at a quarter of the maximal prescribed dose (140 mg/d) [73,74].Our case, however, illustrates that ibrutinib can safely be reintroduced alongside azole-based antifungals under strict monitoring of drug-drug interactions and signs of relapse of the underlying malignancy. ", "section_name": "Management of Cryptococcus gattii Infections in the Haematological Setting", "section_num": "6." }, { "section_content": "The clinical epidemiology, clinical presentation, and course of disease during infections caused by CG seem to involve complex interactions between environmental exposure to CG, infecting genotype, pathogen virulence factors, host susceptibility, and host immune responses.As the spectrum of underlying conditions predisposing individuals to CG infections and the geographical range of CG tend to expand, it is increasingly likely that sporadic cases of CG will be reported in patients with haematological malignancy.It is crucial for future guidelines to address the challenges involved in the management of CG in the onco-haematological setting, regarding both the duration of antifungal induction therapy as well as the management of drug-drug interactions between antifungals and targeted therapies. ", "section_name": "Conclusions", "section_num": "7." } ]	[ { "section_content": "Funding: This research received no external funding. Informed Consent Statement: Patient consent was waived because no personal identifiers were used in the manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "Data Availability Statement: Data supporting the case report are available upon request to the corresponding author. ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare no conflict of interest. ", "section_name": "Conflicts of Interest:", "section_num": null } ]
10.1186/1752-1947-4-246	Cardiac tamponade mimicking tuberculous pericarditis as the initial presentation of chronic lymphocytic leukemia in a 58-year-old woman: a case report	<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Introduction</jats:title> <jats:p>Chronic lymphocytic leukemia is an indolent disease that often presents with complaints of lymphadenopathy or is detected as an incidental laboratory finding. It is rarely considered in the differential diagnosis of patients presenting with tamponade or a large, bloody pericardial effusion. In patients without known cancer, a large, bloody pericardial effusion raises the possibility of tuberculosis, particularly in patients from endemic areas. However, the signs, symptoms and laboratory findings of pericarditis related to chronic lymphocytic leukemia can mimic tuberculosis.</jats:p> </jats:sec> <jats:sec> <jats:title>Case Presentation</jats:title> <jats:p>We report the case of a 58-year-old African American-Nigerian woman with a history of travel to Nigeria and a positive tuberculin skin test who presented with cardiac tamponade. She had a mild fever, lymphocytosis and a bloody pericardial effusion, but cultures and stains were negative for acid-fast bacteria. Assessment of blood by flow cytometry and pericardial biopsy by immunohistochemistry revealed CD5 (+) and CD20 (+) lymphocytes in both tissues, demonstrating this to be an unusual manifestation of early stage chronic lymphocytic leukemia.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Although most malignancies that involve the pericardium clinically manifest elsewhere before presenting with tamponade, this case illustrates the potential for early stage chronic lymphocytic leukemia to present as a large pericardial effusion with tamponade. Moreover, the presentation mimicked tuberculosis. This case also demonstrates that it is possible to treat chronic lymphocytic leukemia-related pericardial tamponade by removal of the fluid without chemotherapy.</jats:p> </jats:sec>	[ { "section_content": "Chronic lymphocytic leukemia (CLL) is an indolent disease that often presents with complaints of lymphadenopathy and fatigue or is detected as an incidental laboratory finding.Although leukemias represent only about eight percent of neoplastic metastases to the heart, almost 50% of lymphoma patients have cardiac involvement at autopsy [1].Pericardial effusions or cardiac tamponade are relatively uncommon as presenting syndromes in patients with hematologic malignancies [2,3].Likewise, hematologic malignancies account for a minority of pericardial effusions.Imazio et al found that only 33 (7.3%) of 450 patients with acute pericardial disease had a neoplastic etiology.The most powerful clinical predictor of a neoplastic etiology was a history of malignancy (odds ratio 19.8) [4].Lung and breast cancers are the most common neoplasms causing pericardial effusion [4].In a series of 150 cases of cardiac tamponade requiring pericardiocentesis, 64% had sanguinous pericardial fluid.The most common causes of the effusions were iatrogenic (31%), followed by malignancy (26%) [5].Only a handful of cases have described cardiac involvement in CLL and these reports described patients with established CLL, rather than patients presenting with tamponade and being subsequently discovered to have CLL [2,[6][7][8].We present the case of a woman whose initial presentation of CLL was cardiac tamponade with sanguinous pericardial fluid but whose history and clinical presentation was suspicious for tuberculous pericarditis. ", "section_name": "Introduction", "section_num": null }, { "section_content": "A 58-year-old African American-Nigerian woman with a week-long history of progressive shortness of breath presented to our emergency room.She had lived in Benin, Nigeria until nine years earlier and had been back for a one-month-long visit three years prior to presentation.She had previously been in excellent health until one week prior to admission.Over the week she became dyspneic with minor exertion and could no longer climb one flight of stairs without pausing to rest.She denied any chest pain, but described a sense of 'congestion' in her chest, which progressed over the week.The patient had a subjective fever that began at the same time as the dyspnea and which was relieved by acetaminophen.Her past medical history was significant for hypertension (treated hydrochlorothiazide and amlodipine) and a positive tuberculin skin test. On physical examination, her vital signs included a heart rate of 100 and blood pressure of 134/94 with a pulsus paradoxus of over 15 mm Hg.Her cardiovascular examination revealed distant heart sounds, normal first and second heart sounds and no murmurs, rubs or knocks.There was jugular venous distention to 20 cm above the manubriosternal angle.The lung examination was unremarkable.Her abdomen was soft and nondistended.There was no peripheral edema, adenopathy or hepatosplemogaly. An initial laboratory assessment was notable for leukocytosis and elevated liver enzymes (Table 1).The chest x-ray revealed cardiomegaly and a small left pleural effusion.The electrocardiogram showed mild QTc prolongation (610 ms) but no signs of low-voltage, electrical alternans or ischemia.The D-dimer level was elevated at 3.16 mg/dl.A multislice computed topography scan, performed to exclude pulmonary embolism, showed no pulmonary embolism, but did reveal mediastinal lymphadenopathy and a large pericardial effusion.Given the her immigrant history and past positive PPD, a working diagnosis of tuberculosis pericarditis was entertained, and she was placed on respiratory isolation. A subsequent transthoracic echocardiogram (Figure 1) showed a large, circumferential pericardial effusion with evidence of right ventricular collapse.Though the her blood pressure remained normal, her jugular venous distention and pulsus paradoxus were consistent with incipient tamponade and pericardiocentesis was performed.Approximately 1L of sanguinous fluid was extracted from the pericardial sac.With drainage the patient immediately improved and had resolution of her dyspnea and normalization of her physical examination. The pericardial fluid contained 2,680,000 red blood cells and 8,500 leukocytes/uL.The leukocyte differential was 33% neutrophils, 56% lymphocytes, 6% macrophages, 2% mesothelial cells and 3% eosinophils.Fluid analysis showed that the fluid glucose was 59 mg/dl, fluid lactate dehydrogenase was 325 IU/L, and total protein was 4.5 g/dl.Staining of the fluid for acid-fast bacilli was negative, as were bacterial cultures.Fluid cytology revealed only reactive mesothelial cells. Because of persistent concern about possible tuberculous pericarditis and lack of a definitive diagnosis, a pericardial window was performed from a subxiphoid approach.On gross examination, the thickened pericardium measured between 0.1 and 0.3 cm.However, acidfast stains and culture remained negative. On the sixth day after admission, the daily complete blood count with differential was notable for the presence of immunoblasts.Flow cytometry of peripheral blood for lymphocyte subsets was performed.A repeat echocardiogram did not demonstrate reaccumulation of pericardial fluid and the patient remained asymptomatic and was discharged home for outpatient evaluation. The flow cytometry results were consistent with CLL.The B/T ratio was 1.8:1.B-cells expressed CD5, CD19, CD20, CD21 (partial), CD22, CD23, CD11c (partial) and CD52, consistent with CLL.Subsequently, histological examination of the pericardium revealed lymphocytic infiltrates surrounding the vascular structures and dispersed within the adipose tissue (Figure 2).The infiltrate was comprised of small lymphocytes with clumped chromatin, indistinct nucleoli and high nuclear/cytoplasmic ratio.Immunohistochemistry of the pericardial tissue demonstrated that the vast majority of the lymphocytes were CD5 (+) (Figure 3) and CD20 (+) B-cells, with a minority of lymphocytes being CD3 (+) T-cells.Together, the flow cytometry of the peripheral blood and immunohistochemistry of the pericardial tissue were consistent with pericardial involvement by CLL. The patient was seen in follow-up at one year and has remained free from symptomatic disease without any chemotherapy.Echocardiography demonstrated that her pericardial effusion had not recurred. ", "section_name": "Case presentation", "section_num": null }, { "section_content": "The etiologies of sanguinous pericardial effusions causing tamponade, excluding iatrogenic causes, include malignancy, renal failure and/or uremia and tuberculosis, although the latter is now uncommon in North America [5].Our initial high suspicion for tuberculous pericarditis was based on her history, the bloody fluid, the mediastinal adenopathy, and the lymphocytosis in the pericardial fluid.An adenosine deaminase assay, which has been used to identify tuberculosis, was uninterpretable due to the amount of blood in the fluid [9].Initially, we discharged the patient under the assumption that the tamponade was most likely due to a viral cause, although Coxsackie and adenoviral titers were negative.It was the late appearance of circulating immunoblasts that finally pointed towards a leukemic process.Up to 20% of patients with a known malignancy are found to have pericardial involvement upon autopsy [10].However, tamponade as an initial manifestation of malignancy is relatively uncommon.A review of 78 cases revealed that 60% of such cases stemmed from lung carcinomas whereas only 9% originated from leukemia or lymphoma [11].The evolution of pericardial effusion is often due to infiltration of malignant cells as well as lymphatic obstruction; our patient clearly had pericardial involvement of leukemia on histology. A PubMed search beginning in 1979 using the keywords \"chronic lymphocytic leukemia and pericarditis, pericardial tamponade, pericardial effusion\" identified only a handful of cases that have documented cardiac infiltration in patients with CLL.One case describes tamponade and pericardial effusion as the initial presentation of lymphosarcoma cell leukemia, which is morphologically very similar to CLL [12].This patient presented with dyspnea and mild abdominal distention and had a leukoctye count of 23,200/uL.Three other cases described tamponade related to previously documented CLL in patients presenting with dyspnea [3,7,8].Finally, one case report detailed constrictive pericarditis in a patient with B-cell chronic lymphatic leukemia whose initial complaint was also breathlessness [6].The leukocyte count in all the CLL patients in these reports was much higher (282,000 to 827,000/uL) than our patient's (18,100/uL). She subsequently continued follow-up with a primary oncologist regarding the status of her CLL.She was found to be Rai stage I and one-year post discharge had not received CLL treatment (because she had normal platelet and hemoglobin levels and remained asymptomatic).Management of pericardial effusions as initial presentations of malignancy is not well established, though some reports have suggested systemic chemotherapy and radiotherapy prior to pericardiocentesis to avoid potential complications [13].In our patient, because the diagnosis of leukemia was unknown, pericardiocentesis was performed without chemotherapy and has provided sustained relief of her dyspnea for the past year. ", "section_name": "Discussion", "section_num": null }, { "section_content": "To the best of our knowledge this is the first reported case of CLL presenting as pericardial tamponade.The diagnosis was confounded by the similarities to tuberculous pericarditis and the modest degree of leukocytosis.The appearance of peripheral immunoblasts was the key to the ultimate diagnosis, which we confirmed by demonstrating CD5 (+) and CD20 (+) lymphocytes using flow cytometry on the blood and immunohistochemistry on the pericardial tissue. ", "section_name": "Conclusion", "section_num": null }, { "section_content": "Written informed consent was obtained from the patient for publication of this case report and accompanying images.A copy of the written consent is available for review by the Editor-in-Chief of this journal. ", "section_name": "Consent", "section_num": null } ]	[ { "section_content": "Authors' contributions EL and SLA were involved in the conception, design, drafting, and revising of the manuscript.All authors were involved in the diagnosis and treatment of the patient and revising the manuscript.All authors read and approved the final manuscript. The authors declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null }, { "section_content": "Authors' contributions EL and SLA were involved in the conception, design, drafting, and revising of the manuscript.All authors were involved in the diagnosis and treatment of the patient and revising the manuscript.All authors read and approved the final manuscript. ", "section_name": "", "section_num": "" }, { "section_content": "The authors declare that they have no competing interests. ", "section_name": "Competing interests", "section_num": null } ]

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